Gamete attachment process revealed in flowering plant fertilization

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule (Fig. 1). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.Open in a separate windowFigure 1.Illustration of the fertilization process in flowering plants. First, each pollen tube accesses an ovule containing egg and central cells. Next, the 2 sperm cells face the female gametes in the ovule after the pollen tube bursts. Finally, each sperm cell simultaneously fuses with either egg or central cell.     

Teaching cases: A patient with loss of vision in the right eye and neurofibromatosis type 1

Abstract: Neurofibromatosis type 1 is a common autosomal dominant condition that affects about 1 in 5000 people. We describe a 75-year-old man who, in addition to many classic developmental changes of the disease in his skin, eyes and nervous system, had blindness in his right eye as a complication.Case: A 75-year-old man with long-standing neurofibromatosis type 1 was admitted because the vision in his right eye had decreased progressively over 3 months. Physical examination showed disseminated cutaneous and subcutaneous neurofibromas of varying size (Figure 1) and café-au-lait spots (Figure 2). The patient had a visual acuity of 6/18 (20/60) in his right eye and Lisch nodules (iris hamartomas) (Figure 3). A neurologic examination showed no abnormalities other than his loss of vision. Axial T₁-weighted magnetic resonance imaging of the brain and orbits (Figure 4) showed an isointense mass lateral to the right optic nerve that appeared atrophic and pushed to the left. The mass showed a hyperintense signal on T₂-weighted images with contrast enhancement. These findings are compatible with glioma of the optic nerve.Open in a separate windowFigure 1: Disseminated cutaneous and subcutaneous neurofibromas of varying size on the torso of a patient with neurofibromatosis type 1.Open in a separate windowFigure 2: A café-au-lait spot on the patient''s right knee.Open in a separate windowFigure 3: Lisch nodules on the left iris.Open in a separate windowFigure 4: T₁-weighted axial magnetic resonance imaging of the brain and orbits, showing an isointense mass lateral to the right optic nerve (white arrow) that appears atrophic and pushed to the left (black arrow on inset).Axial and coronal magnetic resonance imaging (Figure 5) showed a mass in the left parietal lobe with hyperintensity on T₂-weighted images and hypointensity on T₁-weighted images. After a contrast medium was administered, the lesion showed a thickened, enhanced wall with a central necrotic area. These findings are compatible with astrocytoma.Open in a separate windowFigure 5: T₂-weighted axial (left) and coronal (right) magnetic resonance imaging showing a mass with hyperintensity (arrow) in the left temporal lobe. After administration of a contrast medium, the lesion is visible with a thickened enhanced wall and a central necrotic area.Because of slight enlargement and increased hardness of the subcutaneous lesions, an excisional biopsy was performed. Histology showed delicate fascicles consisting of cells with oval or spindle-shaped nuclei, scant cytoplasm and round cells with entrapped axons (Figure 6). Only scattered neoplastic Schwann cells were stained during immunostaining for S-100 protein (Figure 7). This pattern is consistent with neurofibroma. The patient chose not to receive further treatment and was discharged.Open in a separate windowFigure 6: Biopsy specimen of a subcutaneous neurofibroma showing spindle-shaped and round cells with entrapped axons (hematoxylin and eosin, original magnification ×10).Open in a separate windowFigure 7: Only scattered neoplastic Schwann cells (arrow) are stained after immunostaining for S-100 protein. Normally, S-100 protein is present in cells derived from the neural crest, such as Schwann cells. It can be found in melanoma cells, in malignant peripheral nerve sheath tumours and in certain types of sarcomas.Neurofibromatosis type 1, also known as von Recklinghausen disease,¹ is characterized by changes in pigmentation and the growth of tumours along nerves in the skin and other parts of the body. It is caused by a defect in a tumour-suppressing gene on chromosome 17q11.2. Normally the gene produces neurofibromin, a protein that regulates cellular proliferation.² With the gene mutation, the lack of neurofibromin results in overgrowth of cells from neural crest areas in both the central nervous system (causing Schwann cell tumours on virtually every nerve) and the skin. All people who inherit a copy of the mutated gene are affected. As the pattern of inheritance is autosomal dominant, only 1 copy of the defective gene is needed to cause the condition. However, it is not necessary to have an affected parent. About 30%–50% of patients have a new mutation.Neurofibromatosis type 2 is a much rarer form of neurofibromatosis caused by mutations in both alleles of a different tumour suppressor gene on chromosome 22q12.1.About 1 in 3000–5000 individuals are affected by neurofibromatosis type 1, without differences related to ethnic background.³ Pigmented small macules and café-au-lait patches are often present shortly after birth, although neurofibromas are rare in early childhood. In later childhood and adolescence, both neurofibromas and pigmented lesions become common. Clinical manifestations are variable (4Table 1Open in a separate windowA diagnosis of neurofibromatosis type 1 is based on clinical findings. The patient should have 2 or more of the following: 6 or more café-au-lait spots of ≥ 1.5 cm in postpubertal individuals or ≥ 0.5 cm in prepubertal individuals; 2 or more neurofibromas of any type or 1 or more plexiform neurofibroma; and freckling in the underarms and groin.¹ The differential diagnosis includes benign café-au-lait pigmentation (present in up to 10% of the general population), multiple lipomas, and sporadic schwannomas, gliomas and meningiomas in the central nervous system.Most people with mild neurofibromatosis have little disability. People affected by more severe variants have a shortened life expectancy, especially if tumours of the central nervous system or other malignant neoplasms arise during the course of illness.^1,3 The condition can have a serious psychological impact because the accumulation of skin nodules can be quite disfiguring.⁵ Surgical excision and laser treatment of the neurofibromas are possible, but neither treatment is universally effective.⁶ Transplantation with an allograft of composite tissue on the lower and middle parts of a patient''s face was recently reported.⁷Gliomas of the optic nerve are found in up to 15% of pediatric patients with neurofibromatosis type 1. Best detected using magnetic resonance imaging, these gliomas are symptomatic in about 50% of patients at diagnosis. A minority will progress to vision loss.⁸ The high prevalence of gliomas of the optic nerve that are asymptomatic may, however, be biased by referral patterns, Indeed, in patients with neurofibromatosis type 1, the threshold of risk for optic nerve glioma is low.⁹Guidelines are available for the diagnosis and management of neurofibromatosis type 1.^10,11 Physicians who identify patients with neurofibromatosis type 1 should refer them early to facilities where appropriate evaluation and monitoring of lesions can be carried out. Early detection and monitoring may help to prevent disability and death.     

Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel Cell Polyomavirus

The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its entry mechanism and pathophysiology.Members of the polyomavirus (PyV) family, including simian virus 40 (SV40), murine PyV (mPyV), and BK virus (BKV), bind cell surface gangliosides to initiate infection (2, 8, 11, 15). PyV capsids are assembled from 72 pentamers (capsomeres) of the major coat protein VP1, with the internal proteins VP2 and VP3 buried within the capsids (7, 12). The VP1 pentamer makes direct contact with the carbohydrate portion of the ganglioside (10, 12, 13) and dictates the specificity of virus interaction with the cell. Gangliosides are glycolipids that contain a ceramide domain inserted into the plasma membrane and a carbohydrate domain that directly binds the virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15), mPyV binds to gangliosides GD1a and GT1b (11, 15), and BKV binds to gangliosides GD1b and GT1b (8).Recently, a new human PyV designated Merkel cell PyV (MCPyV) was identified in Merkel cell carcinomas, a rare but aggressive skin cancer of neuroendocrine origin (3). It is as yet unclear whether MCPyV is the causative agent of Merkel cell carcinomas (17). A key to understanding the infectious and transforming properties of MCPyV is the elucidation of its cellular entry pathway. In this study, we identify a putative host cell receptor for MCPyV.Because an intact infectious MCPyV has not yet been isolated, we generated recombinant MCPyV VP1 pentamers in order to characterize cellular factors that bind to MCPyV. VP1 capsomeres have been previously shown to be equivalent to virus with respect to hemagglutination properties (4, 16), and the atomic structure of VP1 bound to sialyllactose has demonstrated that the capsomere is sufficient for this interaction (12, 13). The MCPyV VP1 protein (strain w162) was expressed and purified as described previously (1, 6). Briefly, a glutathione S-transferase-MCPyV VP1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose affinity chromatography. The fusion protein was eluted using glutathione and cleaved in solution with thrombin. The thrombin-cleaved sample was then rechromatographed on a second glutathione-Sepharose column to remove glutathione transferase and any uncleaved protein. The unbound VP1 was then chromatographed on a P-11 phosphocellulose column, and peak fractions eluting between 400 and 450 mM NaCl were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining (Fig. ​(Fig.1A,1A, left) and immunoblotting using an antibody (I58) that generally recognizes PyV VP1 proteins (Fig. ​(Fig.1A,1A, right) (9). Transmission electron microscopy (Philips CM10) analysis confirmed that the purified recombinant MCPyV VP1 formed pentamers (capsomeres), which did not assemble further into virus-like particles (Fig. ​(Fig.1B).1B). In an initial screening of its cell binding properties, we tested whether the MCPyV VP1 pentamers hemagglutinated red blood cells (RBCs). The MCPyV VP1 pentamers were incubated with sheep RBCs and assayed as previously described (5). SV40 and mPyV recombinant VP1 pentamers served as negative and positive controls, respectively. We found that MCPyV VP1 hemagglutinated the RBCs with the same efficiency as mPyV VP1 (protein concentration/hemagglutination unit) (Fig. ​(Fig.1C,1C, compare rows B and C from wells 1 to 11), suggesting that MCPyV VP1 engages a plasma membrane receptor on the RBCs. The recombinant murine VP1 protein used for comparison was from the RA strain, a small plaque virus (4). Thus, MCPyV VP1 has the hemagglutination characteristics of a small plaque mPyV (12, 13).Open in a separate windowFIG. 1.Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated. (B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 μg/ml and absorbed onto Formvar/carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission electron microscopy at 80 kV. Bar = 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40 VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 μg/ml (well 1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 μg/ml VP1 protein.To characterize the chemical nature of the putative receptor for MCPyV, total membranes from RBCs were purified as described previously (15). The plasma membranes (30 μg) were incubated with MCPyV VP1 (0.5 μg) and floated on a discontinuous sucrose gradient (15). After fractionation, the samples were analyzed by SDS-PAGE, followed by immunoblotting with I58. VP1 was found in the bottom of the gradient in the absence of the plasma membranes (Fig. ​(Fig.2A,2A, first panel). In the presence of plasma membranes, a fraction of the VP1 floated to the middle of the gradient (Fig. ​(Fig.2A,2A, second panel), supporting the hemagglutination results that suggested that MCPyV VP1 binds to a receptor on the plasma membrane.Open in a separate windowFIG. 2.MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with proteinase K (4 μg/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with or without α2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.To determine whether the receptor is a protein or a lipid, plasma membrane preparations (30 μg) were incubated with proteinase K (Sigma), followed by analysis with SDS-PAGE and Coomassie blue staining. Under these conditions, the majority of the proteins in the plasma membranes were degraded by the protease (Fig. ​(Fig.2B,2B, compare lanes 1 and 2). Despite the lack of proteins, the proteinase K-treated plasma membranes bound MCPyV VP1 as efficiently as control plasma membranes (Fig. ​(Fig.2A,2A, compare the second and third panels), demonstrating that MCPyV VP1 interacts with a protease-resistant receptor. The absence of VP1 in the bottom fraction in Fig. ​Fig.2A2A (third panel) is consistent with the fact that the buoyant density of the membranes is lowered by proteolysis. Of note, a similar result was seen with binding of the mPyV to the plasma membrane (15). Binding of MCPyV to the cell surface of two human tissue culture cells (i.e., HeLa and 293T) was also largely unaffected by pretreatment of the cells with proteinase K (Fig. 2C and D, compare lanes 1 and 2), further indicating that a nonproteinaceous molecule on the plasma membrane engages the virus.We next determined whether the protease-resistant receptor contains a sialic acid modification. Plasma membranes (10 μg) were incubated with a neuraminidase (α2-3,6,8 neuraminidase; Calbiochem) to remove the sialic acid groups. In contrast to the control plasma membranes, the neuraminidase-treated membranes did not bind MCPyV VP1 (Fig. ​(Fig.2E,2E, compare first and second panels), indicating that the MCPyV receptor includes a sialic acid modification.Gangliosides are lipids that contain sialic acid modifications. We asked if MCPyV VP1 binds to gangliosides similar to other PyV family members. The structures of the gangliosides used in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b) are depicted in Fig. ​Fig.3A.3A. To assess a possible ganglioside-VP1 interaction, we employed a liposome flotation assay established previously (15). When liposomes (consisting of phosphatidyl-choline [19 μl of 10 mg/ml], -ethanolamine [5 μl of 10 mg/ml], -serine [1 μl of 10 mg/ml], and -inositol [3 μl of 10 mg/ml]) were incubated with MCPyV VP1 and subjected to the sucrose flotation assay, the VP1 remained in the bottom fraction (Fig. ​(Fig.3B,3B, first panel), indicating that VP1 does not interact with these phospholipids. However, when liposomes containing GT1b (1 μl of 1 mM), but not GM1 (1 μl of 1 mM) or GD1a (1 μl of 1 mM), were incubated with MCPyV VP1, the vesicles bound this VP1 (Fig. ​(Fig.3B).3B). A low level of virus floated partially when incubated with liposomes containing GD1b (Fig. ​(Fig.3B),3B), perhaps reflecting a weak affinity between MCPyV and GD1b. Importantly, MCPyV binds less efficiently to neuraminidase-treated GT1b-containing liposomes than to GT1b-containing liposomes (Fig. ​(Fig.3B,3B, sixth panel), suggesting that the GT1b sialic acids are involved in virus binding. This finding is consistent with the ability of neuraminidase to block MCPyV binding to the plasma membrane (Fig. ​(Fig.2E).2E). The level of virus flotation observed in the neuraminidase-treated GT1b-containing liposomes is likely due to the inefficiency of the neuraminidase reaction with a high concentration of GT1b used to prepare the vesicles.Open in a separate windowFIG. 3.MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides. The samples were analyzed as described in the legend to Fig. ​Fig.2A.2A. Where indicated, GT1b-containing liposomes were pretreated with α2-3,6,8 neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as described in the legend to panel B.As controls, GM1-containing liposomes bound SV40 (Fig. ​(Fig.3C),3C), GD1a-containing liposomes bound mPyV (Fig. ​(Fig.3D),3D), and GD1b-containing liposomes bound BKV (Fig. ​(Fig.3E),3E), demonstrating that the liposomes were functionally intact. We note that, while all of the MCPyV VP1 floated when incubated with liposomes containing GT1b (Fig. ​(Fig.3B,3B, sixth panel), a significant fraction of SV40, mPyV, and BKV VP1 remained in the bottom fraction despite being incubated with liposomes containing their respective ganglioside receptors (Fig. 3C to E, second panels). This result is likely due to the fact that in contrast to MCPyV, which are assembled as pentamers (Fig. ​(Fig.1B),1B), the SV40, mPyV, and BKV used in these experiments are fully assembled particles: their larger and denser nature prevents efficient flotation. Nonetheless, we conclude that MCPyV VP1 binds to ganglioside GT1b efficiently.The observation that GD1a does not bind to MCPyV VP1 suggests that the monosialic acid modification on the right branch of GT1b (Fig. ​(Fig.3A)3A) is insufficient for binding. Similarly, the failure of GD1b to bind MCPyV VP1 suggests that the sialic acid on the left arm of GT1b is necessary for binding. Together, these observations suggest that MCPyV VP1 interacts with sialic acids on both branches of GT1b (Fig. ​(Fig.4).4). A recent structure of SV40 VP1 in complex with the sugar portion of GM1 (10) demonstrated that although SV40 VP1 binds both branches of GM1 (Fig. ​(Fig.4),4), only a single sialic acid in GM1 is involved in this interaction. In the case of mPyV, structures of mPyV VP1 in complex with different carbohydrates (12, 13) revealed that the sialic acid-galactose moiety on the left branch of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig. ​(Fig.4).4). Although no structure of BKV in complex with the sugar portion of GD1b (or GT1b) is available, in vitro binding studies (8) have suggested that the disialic acid modification on the right branch of GD1b (and GT1b) is responsible for binding BKV VP1 (Fig. ​(Fig.4).4). Thus, it appears that the unique feature of the MCPyV VP1-GT1b interaction is that the sialic acids on both branches of this ganglioside are likely involved in capsid binding.Open in a separate windowFIG. 4.A potential model of the different VP1-ganglioside interactions (see the text for discussion).The identification of a potential cellular receptor for MCPyV will facilitate the study of its entry mechanism. An important issue for further study is to determine whether MCPyV targets Merkel cells preferentially, and if so, whether GT1b is found in higher levels in these cells to increase susceptibility.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

Teaching cases: A 1-week-old newborn with hypercalcemia and palpable nodules: subcutaneous fat necrosis

Abstract: We present a 1-week-old newborn with subcutaneous fat necrosis complicated by hypercalcemia. She received conservative treatment of adequate hydration and restricted supplementary vitamin D.The case: A 1-week-old term newborn girl was brought to her physician with a 2-day history of subcutaneous masses. The girl had been born by vacuum-assisted vaginal delivery with a birth weight of 3.5 kg. She did not require resuscitation but was observed for 24 hours in a special care nursery because of tachypnea. The patient was discharged home after 48 hours, and her course over the next 5 days was unremarkable.On physical examination, the newborn was afebrile and in no obvious distress. She had multiple firm, mobile, mildly tender subcutaneous nodules with overlying erythema (Figure 1). The largest mass on palpation was located in the left deltoid area and measured 2 × 2.5 cm (Figure 1). Two smaller lesions were located in the left posterior axillary area (Figure 1) and a fourth lesion was in the right posterior auricular region. The remainder of the physical examination and the results of a complete blood count were normal. The newborn''s total serum calcium level was elevated (2.94 [normal 1.96–2.66] mmol/L) as was her ionized calcium level (1.36 [normal 1.14–1.29] mmol/L. A biopsy was not performed because the infant was well and the results of clinical investigations were consistent with subcutaneous fat necrosis.Open in a separate windowFigure 1: A 5-day-old infant with lesions of subcutaneous fat necrosis. The largest mass (black arrow) measured 2 × 2.5 cm. Two smaller lesions (white arrows) were located in the left posterior axillary area.Subcutaneous fat necrosis of the newborn is a relatively uncommon condition that occurs in the first several weeks after birth. The incidence is unknown; however, it is more frequently reported after perinatal distress than after uncomplicated deliveries, and maternal risk factors include gestational diabetes and preeclampsia.¹ Skin lesions are characterized by indurated nodules that range from flesh-coloured to blue and by plaques on the face, trunk and buttocks as well as on the arms and legs near the trunk. Figure 2 is a representative microscopic image of this condition.² The differential diagnosis is bacterial cellulitis, erysipelas and sclerema neonatorum.³Open in a separate windowFigure 2: Left: A typical photomicrograph from a different patient showing lobular panniculitis with sparing of the dermis and epidermis (original magnification × 20). Right: A high-power view shows that the inflammatory infiltrate is mixed and composed of histiocytes, lymphocytes, neutrophils and eosinophils. Cleft-like spaces (arrow) suggestive of dissolved crystals can be seen at the periphery of some of the fat cysts (original magnification × 400). Reproduced with permission from Macmillan Publishers Ltd: Journal of Perinatology (Diamantis et al.²) © 2006.Although subcutaneous fat necrosis of the newborn is often benign and self-limited, the most important concern is hypercalcemia, which can lead to neurologic or cardiac problems, nephrocalcinosis and nephrolithiasis. Clinical signs of newborn hypercalcemia include irritability, poor feeding and vomiting. Skin lesions typically resolve over a period of weeks to several months; however, hypercalcemia can persist longer and requires serial monitoring. The treatment of hypercalcemia ranges from conservative measures such as hydration and restriction of vitamin D and calcium to more aggressive interventions such as furosemide, glucocorticoid or bisphosphonate therapy in severe cases.⁴In our patient, mild hypercalcemia was accompanied by mild elevations in the ratio of calcium to creatinine in the urine and a normal 1,25-dihydroxyvitamin D level. Because our patient was otherwise well, we opted for conservative management. In 2 months, her calcium level had normalized and the lesions completely regressed.Fahed Aljaser MD Michael Weinstein MD Division of Pediatric Medicine Hospital for Sick Children University of Toronto Toronto, Ont.     

The hyponatremic patient: a systematic approach to laboratory diagnosis

HYPONATREMIA (SERUM SODIUM LEVEL LESS THAN 134 MMOL/L) is a common electrolyte disturbance. Its high prevalence and potential neurologic sequelae make a logical and rigorous differential diagnosis mandatory before any therapeutic intervention. A history of concurrent illness and medication use as well as the assessment of extracellular volume status on physical examination may provide useful clues as to the pathogenesis of hyponatremia. Measurement of the effective serum tonicity (serum osmolality less serum urea level) is the first step in the laboratory evaluation. In patients with normal or elevated effective serum osmolality (280 mOsm/kg or greater), pseudohyponatremia should be excluded. In the hypo-osmolar state (serum osmolality less than 280 mOsm/kg), urine osmolality is used to determine whether water excretion is normal or impaired. A urine osmolality value of less than 100 mOsm/kg indicates complete and appropriate suppression of antidiuretic hormone secretion. A urine sodium level less than 20 mmol/L is indicative of hypovolemia, whereas a level greater than 40 mmol/L is suggestive of the syndrome of inappropriate antidiuretic hormone secretion. Levels of hormones (thyroid-stimulating hormone and cortisol) and arterial blood gases should be determined in difficult cases of hyponatremia.Hyponatremia (serum sodium level less than 134 mmol/L) is a common electrolyte disturbance occurring in a broad spectrum of patients, from asymptomatic to critically ill.^1,2 There are serious neurologic sequelae associated with hyponatremia and its treatment. Therefore, a logical, rigorous differential diagnosis is mandatory before therapy can be begun.^3,4 Since hyponatremia is caused primarily by the retention of solute-free water, its cause encompasses disorders associated with limitation in water excretion.⁵ The principal causes of hyponatremia are summarized in Open in a separate windowAs with other electrolyte abnormalities, the history and physical examination can provide important clues toward the correct diagnosis. In most cases the initial laboratory evaluation includes measurement of serum osmolality and urine osmolality (by osmometer if available), urine sodium concentration and serum levels of other electrolytes (potassium, chloride and bicarbonate) as well as serum concentrations of urea, glucose, uric acid, total proteins and triglycerides. In addition, determination of serum levels of thyroid-stimulating hormone and cortisol is important to exclude any associated endocrinopathy (Fig. 1). Measurement of arterial blood gases is also useful in the differential diagnosis of hyponatremia, particularly in patients with abnormal serum bicarbonate concentrations.Open in a separate windowFig. 1: Clinical diagnostic algorithm for hyponatremia. TSH = thyroid-stimulating hormone, EABV = effective arterial blood volume, SIADH = syndrome of inappropriate secretion of antidiuretic hormone, FE = fractional excretion.Table 2Open in a separate windowThe step-by-step diagnostic evaluation of hyponatremia is shown in Fig. 1.     

Simple,Fast, and Sensitive Method for Quantification of Tellurite in Culture Media

A fast, simple, and reliable chemical method for tellurite quantification is described. The procedure is based on the NaBH₄-mediated reduction of TeO₃²⁻ followed by the spectrophotometric determination of elemental tellurium in solution. The method is highly reproducible, is stable at different pH values, and exhibits linearity over a broad range of tellurite concentrations.The tellurium oxyanion tellurite is toxic for most organisms, making important its accurate assessment. Several methods for quantifying tellurite have been described to date. However, most of them are rather complicated and require sophisticated equipment and in some cases the detection is not quite sensitive enough to allow the assessment of TeO₃²⁻ concentrations below 50 μg/ml (200 μM). For example, the analytical determination of tellurium (Te) oxyanions by atomic absorption spectrometry (AAS) is hampered by poor sensitivity. Where flame or electrothermal AAS routinely yields detection limits of less than 10 ppb for iron (16), normal flame AAS tellurium detection limits are 100 to 1,000 times higher and require pretreatment to achieve the +IV oxidation state before analysis (11).On the other hand, hydride generation AAS (HGAAS) is used to achieve ppb-level detection limits for Se and Te as well as arsenic and antimony among others. For Te the volatile hydride gas H₂Te is generated by first converting the metalloid to the +IV oxidation state and then by chemical reduction to the gaseous hydride using—almost universally—sodium borohydride (NaBH₄). In automated HGAAS systems, an inert purge gas sweeps the volatile hydride formed in a glass reaction vessel into a quartz cell heated by the AAS flame where gaseous hydride decomposition and atomization occur. Though tellurite reduction, precipitation, and detection methods have been reported (3, 17), they are temporally relatively unstable and pH dependent.Since tellurium is toxic and environmentally important (7, 8), determining low concentrations in bacterial cultures is very desirable and a simple analysis without pretreatment steps that could quickly establish total metalloid oxyanion content in a liquid sample would be a plus. Here we report a new method for the determination of tellurite in bacterial culture media. This procedure is based on the NaBH₄ reduction of tellurite to the elemental form, which is analyzed spectrophotometrically at 500 nm or 320 nm (see below), by which the light scattered by the particles of elemental metalloid in solution is measured. While the detection limits do not compare to those of HGAAS (14) or capillary electrophoresis (13), they do approach those of old flame AAS but involve a much simpler and quicker procedure requiring only one reagent and a spectrophotometer to determine total content of solutions of +IV oxyanions in solution. Linear calibration range, method development time and probe stability, effect of sample pH, common interferences, and detection limits were investigated.Calibration curves to determine K₂TeO₃ concentrations in routinely used microbiological culture media such as Luria-Bertani (LB) or M9 minimal medium amended with 0.2% glucose (15) were constructed. A set of solutions containing increasing concentrations of K₂TeO₃ (Sigma) were prepared in LB or M9 culture medium, and the tellurium oxyanion was quantitatively reduced using freshly prepared 3.5 mM NaBH₄ (final concentration).The reaction was carried out at 60°C for 10 min (bubbling was overcome by vortexing), and after 5 min at room temperature, the optical density at 500 nm (OD₅₀₀) was determined spectrophotometrically as described previously (4, 5, 9, 12). Blanks contained no borohydride. Figure ​Figure11 shows that in both media good curve linearity was obtained, with r² values of 0.9740 and 0.9963 for LB and M9, respectively. Tellurite concentrations lower than 1 μg/ml or higher than 200 μg/ml were also tested, but OD₅₀₀ values were close to the spectrophotometer error limit at low concentrations or nonlinear above 200 μg/ml (not shown). Thus, the NaBH₄ method allows determination of a wide range of tellurite concentrations in a fast and simple way. Tellurite concentrations lower than 50 μg/ml in both rich and minimal media can be easily determined; the experimental error was about 10%, similar to that reported for the diethyl dithiocarbamate (DDTC) tellurite method (17).Open in a separate windowFIG. 1.Calibration curves to determine K₂TeO₃ concentrations in LB (A) (R² = 0.9963) or M9 minimal (B) (R² = 0.9740) medium. Optical density at 500 nm was determined after reducing the oxyanion with sodium borohydride. Error bars denote 1 standard deviation of three replicates.To analyze the resulting solutions after tellurite reduction by NaBH₄, absorption/scattering spectra were determined. Figure ​Figure22 shows that spectra from LB and those from M9 after tellurite reduction are quite different, which may be a consequence of the different chemical compositions of these culture media. In both cases, absorption spectra showed linearity between optical density at 500 nm and tellurite concentration in the sample. However, high tellurite concentrations (∼100 μg/ml) caused a loss of linearity in LB medium.Open in a separate windowFIG. 2.Absorption spectra after reducing samples of LB (A) or M9 (B) culture medium containing increasing tellurite concentrations with 3.5 mM NaBH₄. Tellurite concentrations used were 20, 40, 60, 80, and 100 (LB) and 2, 4, 6, 8, and 10 (M9) μg/ml. (Inset) Calibration curve in M9 medium using the absorbance maxima at 320 nm.Figure ​Figure2B2B shows that in M9 medium there is a zone around 320 nm exhibiting higher optical density than that at 500 nm, which represents an advantage in the determination of tellurite in chemically defined culture media. This is reflected in a wider range of measurable concentrations at 320 nm (Fig. ​(Fig.2B,2B, inset), as well as in a higher sensitivity of the method as determined by the slope of the calibration curve. The product of tellurite reduction by NaBH₄ showed good stability at both wavelengths in rich and minimal culture media (not shown).Since in M9 medium the method allows the determination of minor tellurite concentrations (1 to 20 μg/ml), it would be of great help in assessing tellurite uptake in tellurite-sensitive microorganisms whose MICs range from 1 to 10 μg/ml. Sulfur-containing salts, commonly present in culture media as sulfites and sulfates, did not interfere with our NaBH₄ method for tellurite assessment at concentrations up to 0.5 M (not shown).As shown in Fig. ​Fig.3,3, tellurite assessment was not affected by the pH of the culture medium. In fact, linearity was observed in a wide pH range with minor slope changes in LB. Similar results were obtained with M9 medium, although tellurite assessment was not possible at pH values higher than 7.0 because of the formation of a precipitate. This may be due to an interaction of the phosphate salts present in the medium and some charged (2⁺) chemical species forming at alkaline pH values, as has been reported earlier (17).Open in a separate windowFIG. 3.Effect of pH in determining tellurite concentrations in LB (A) and M9 minimal (B) media.To date, the most commonly used procedure for determining tellurite in culture media is that involving the spectrophotometric determination (340 nm) of the complex that forms between tellurite and diethyl dithiocarbamate (17). This procedure has been used to assess tellurite uptake by the phototrophic bacterium Rhodobacter capsulatus, which is naturally resistant to K₂TeO₃ (MIC, ∼1.4 mM) (2, 3). However, K₂TeO₃ uptake studies in highly sensitive cells such as Escherichia coli (MIC, ∼4 μM) are difficult to carry out because of the low concentrations of toxicant present in the culture medium, far below the detection limit of the DDTC procedure (17).In this context and for testing the applicability of our method in vivo, we used the tellurite-sensitive bacterium E. coli BW25113 (10) and the tellurite-resistant Aeromonas caviae ST (5, 6). An overnight culture of E. coli BW25113 in M9 minimal medium was diluted 100-fold with fresh M9 supplemented with 0.2% glucose and grown at 37°C with shaking. When the OD₆₀₀ was 0.1, the culture was amended with 20 μg/ml K₂TeO₃ (arrow, Fig. ​Fig.4A).4A). Then aliquots were taken at the indicated times and cells were centrifuged at 8,500 × g for 3 min; supernatants were used to assess extracellular tellurite by our NaBH₄ method. While added tellurite did not affect bacterial growth (Fig. ​(Fig.4A),4A), the remaining tellurite in the supernatant dropped approximately to one-third after 3 h (Fig. ​(Fig.4B).4B). Tellurite determinations were validated using, in parallel, the DDTC method (not shown).Open in a separate windowFIG. 4.Tellurite uptake by Escherichia coli. Time zero in panel B represents the moment of tellurite addition.Regarding the tellurite-resistant bacterium A. caviae ST, a 1:100 dilution of an overnight culture was inoculated into fresh LB medium and the OD₆₀₀ was recorded at the indicated times. When the OD₆₀₀ was ∼0.4, the culture was amended with tellurite (400 μg/ml final concentration) (Fig. ​(Fig.5A,5A, arrow) and the remaining tellurite in the supernatants was assessed as described above. Figure ​Figure5B5B shows that in 4 h ∼27% of the toxic oxyanion was removed from the culture medium.Open in a separate windowFIG. 5.Tellurite uptake by Aeromonas caviae ST. See the text for details.In summary and in comparison to the DDTC procedure, the NaBH₄ method described here allows more sensitive determination of the initial tellurite concentrations as well as the continuous uptake of the toxicant by tellurite-sensitive and tellurite-resistant microorganisms. This method should be of great help in future studies aimed at unveiling the tellurite reductase activity exhibited by some metabolic enzymes such as nitrate reductase (1), catalase (4), and the pyruvate dehydrogenase complex (5, 6). These studies are currently being carried out in our laboratory.     

Hepatitis C Virus Egress and Release Depend on Endosomal Trafficking of Core Protein

Hepatitis C virus (HCV) assembly is known to occur in juxtaposition to lipid droplets, but the mechanisms of nascent virion transport and release remain poorly understood. Here we demonstrate that HCV core protein targets to early and late endosomes but not to mitochondria or peroxisomes. Further, by employing inhibitors of early and late endosome motility in HCV-infected cells, we demonstrate that the movement of core protein to the early and late endosomes and virus production require an endosome-based secretory pathway. We also observed that this way is independent of that of the internalization of endocytosed virus particles during virus entry.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV usually infects host cells via receptor-mediated endocytosis (6, 21), followed by the release of genomic RNA after uncoating of the nucleocapsid in the endosome. HCV core protein constitutes the viral nucleocapsid and may possess multiple functions. Intracellular HCV core protein is localized mainly in lipid droplets (LDs) (23, 29). Recent studies have indicated that core protein promotes the accumulation of LDs to facilitate virus assembly (1, 10) and recruits viral replication complexes to LD-associated membranes, where virus assembly takes place (23). However, the precise mechanisms of HCV assembly, budding, and release remain largely unclear. Most recently, HCV virion release has been shown to require the functional endosomal sorting complex required for transport III (ESCRT-III) and Vps4 (an AAA ATPase) (13), which are required for the biogenesis of the multivesicular body (MVB), a late endosomal compartment (12). Late endosomes have been implicated in the budding of several other viruses, including retroviruses (8, 17, 24, 25, 27), rhabdoviruses (14), filoviruses (18, 20), arenaviruses (26, 32), and hepatitis B virus (35). However, little is known about the roles of late endosomes in the HCV life cycle.Since LDs are associated with the endoplasmic reticulum membrane, endosomes, peroxisomes, and mitochondria (16, 37), we investigated what subcellular compartments may be involved in HCV assembly and release. We first compared the intracellular distribution of HCV core protein with that of early endosome markers Rab5a and early endosome antigen 1 (EEA1), as well as the late endosome marker CD63 in the HCV Jc1-infected Huh7.5 cells at day 10 postinfection (p.i.). In immunofluorescence studies, we demonstrated that the core protein partially colocalized with Rab5a (Fig. ​(Fig.1A,1A, left panel) or EEA1 (Fig. ​(Fig.1A,1A, right panel). This finding was confirmed by the expression of enhanced green fluorescent protein (EGFP)-tagged Rab5a (Fig. ​(Fig.1A,1A, middle panel). Similarly, core protein also showed partial colocalization with CD63 (Fig. ​(Fig.1B).1B). In particular, core protein showed numerous vesicle-like structures of homogeneous size that partially colocalized with CD63 at the cell periphery (Fig. ​(Fig.1B,1B, right panel inset and drawing). This result contrasts with that of Ai et al. (2), who observed, by confocal microscopy, that core protein did not interact with markers of early and late endosomes. Ai et al. did find, however, that multimeric core complexes cofractionated with ER/late endosomal membranes in HCV-infected cells.Open in a separate windowFIG. 1.HCV core protein colocalized with early and late endosomes but not mitochondria and peroxisomes. HCV-infected cells were costained with anti-core protein (red) and anti-Rab5a (A, left panel), -EEA1 (A, right panel), or -CD63 antibodies (green) (B) or transfected with plasmids expressing enhanced green fluorescent protein (EGFP)-Rab5a (A, middle panel), enhanced yellow fluorescent protein (EYFP) (C, left panel), EYFP-mitochondria (C, middle panel), or EYFP-peroxisome (C, right panel). Cellular DNA was labeled with DAPI (4′,6-diamidine-2-phenylindole) (blue). Images shown were collected sequentially with a confocal laser scanning microscope and merged to demonstrate colocalization (yellow merge fluorescence). Enlarged views of parts of every image are shown (insets). The cartoon in panel B illustrates the core protein-containing vesicle-like structures (depicted as red circles), which partially colocalized with CD63 at the cell periphery in HCV-infected cells. PM, plasma membrane.To demonstrate the specificity of the association of core protein with endosomes, we transfected HCV-infected cells (at day 10 p.i.) with pEYFP, pEYFP-mito, and pEYFP-peroxi (Clontech) (Fig. ​(Fig.1C),1C), which label the cytoplasm/nucleus, mitochondria, and peroxisomes, respectively. The results showed that HCV core protein did not colocalize with mitochondria or peroxisomes. Taken together, these results indicate that core protein is partially associated with early and late endosomes.To investigate the functional involvement of the endosomes in HCV release, we employed HCV-infected cells (at day 10 p.i.). In our observation, at day 10 p.i., not all the cells were infected with HCV, as revealed by immunofluorescence staining against core protein (data not shown), suggesting that these cells are a mixture of infected and noninfected cells. We examined the effects of inhibitors of endosome movement, including 10 μM nocodazole (which induces microtubule depolymerization), 100 nM wortmannin (which inhibits early endosomes), 20 nM Baf-A1 (which blocks early endosomes from fusing with late endosomes) (Sigma), and 10 μg/ml U18666A (which arrests late endosome movement) (Biomol), on the release of HCV in the HCV-infected cells. We first determined the possible cytotoxicity of these drugs. We found that within 20 h of the drug application, no significant effect on cell viability, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, was observed (Fig. ​(Fig.2C).2C). We therefore treated the cells with the various drugs for 20 h. This protocol focuses only on virus release, not on virus entry, as the reinfection of Huh7.5 cells could not account for the effects on virus release, as one round of HCV replication requires about 24 h (11).Open in a separate windowFIG. 2.HCV virion release requires endosome motility. HCV-infected cells (at day 10 p.i.) were treated with DMSO, nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) for 20 h, and then the levels of extracellular (A) and intracellular (B) HCV RNA and HCV core proteins (F) in cells were analyzed by RT-qPCR and Western blotting, respectively. Results of Western blotting were quantified by PhosphorImager counting. (C) Analysis of cellular proliferation and survival by MTS assay. (D) Assay of extracellular viral infectivity. The culture supernatants from the cells treated with the various drugs as indicated were used to infect naïve Huh7.5 cells. The cells were stained with anti-core protein antibody (green) and DAPI (blue). The images were analyzed by using Metamorph, and the proportion of cells (of 5,000 counted) expressing core protein was counted (E). Results are presented as percentages and are averages and standard deviations from results of triplicate experiments. (G) In parallel, the HCV-infected cells were costained with anti-Rab5a (green) and -NS5A (red) antibodies. Cellular DNA was labeled with DAPI (blue). Enlarged views of parts of every image are shown (insets). (H) Colocalization efficiency between NS5A and early endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. (I) Assay of intracellular HCV titers. Intracellular HCV particles were prepared from the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 10 μg/ml for 20 h. The titers of intracellular HCV particles were determined by immunofluorescence staining for core-positive cell foci and are reported in focus-forming units (FFU)/ml. (J) HCV-infected cells (at day 10 p.i.) were treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h, and then the levels of extracellular HCV RNA were analyzed by RT-qPCR. (K) Effects of nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) on HCV production in single-cycle HCV growth assays. Huh7.5 cells were infected with HCV at an MOI of 1 and then incubated with DMEM containing the various drugs. At 24 h p.i., the cells and their culture supernatants were collected and used to determine the levels of intracellular and extracellular HCV RNA, which were converted to percentages of the control levels (DMSO) as 100%. Noc, nocodazole; U18, U18666A; Baf-A1, Bafilomycin A1; Wortman, wortmannin.After treatments for 20 h, the cells and their culture supernatants were collected. Intracellular RNA was isolated from cell lysates using a High Pure RNA isolation kit (Roche), and viral RNA was isolated from cell culture supernatants using a QIAamp viral RNA kit (Qiagen). Equivalent RNA volumes were subsequently analyzed on a LightCycler 1.5 real-time PCR system (Roche) for quantitative PCR (qPCR), with a primer-probe set specific for the 5′ untranslated region (UTR) sequence of HCV Jc1 and a second set specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene to quantitate the RNA amount. To calculate the percentage of HCV RNA remaining after the inhibitor treatments, the mean HCV RNA levels from triplicate wells of each sample type were standardized to the mean GAPDH RNA level in the dimethyl sulfoxide (DMSO) control wells. The relative levels of HCV RNA (percentage of control) were then analyzed with LightCycler software (version 3.53) and calculated using the relative quantification method as described in Roche Applied Science, Relative Quantification, Technical Note no. LC 13/2001 (http://www.gene-quantification.de/roche-rel-quant.pdf).As indicated in Fig. ​Fig.2A,2A, the extracellular HCV RNA levels were reduced to 32.1%, 21.7%, 76.2%, and 53.8% of the DMSO control after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 64% by nocodazole treatment (Fig. ​(Fig.2B),2B), confirming previous reports that microtubules are required for HCV RNA synthesis (9, 28). Treatments with Baf-A1 and wortmannin, however, did not affect HCV RNA replication. Interestingly, the intracellular HCV RNA level was increased to 167% by U18666A treatment (Fig. ​(Fig.2B),2B), suggesting that U18666A blocks HCV particle release (Fig. ​(Fig.2A),2A), thereby causing accumulation of HCV RNA in the late endosomes. We further determined the extracellular viral infectivity after treatments with these drugs by using the culture supernatant to infect naïve Huh7.5 cells. The infectivity was checked by counting core protein-expressing cells. Raw acquired 8-bit images (Fig. ​(Fig.2D)2D) were converted to 16-bit and analyzed with the Multi Wavelength Cell Scoring application module in Metamorph (Molecular Device). All of these treatments significantly inhibited the production of infectious HCV particles, as shown by the reduced infectivity in the supernatant of the infected cells (Fig. 2D and E). Determination of the amounts of core protein in the lysates showed that the levels of core protein in cells were not significantly affected by the various drug treatments (Fig. ​(Fig.2F).2F). Taken together, these results suggest that the inhibitors of endosome movement, including U18666A, Baf-A1, and wortmannin, reduced the secretion of HCV particles but not the HCV RNA replication. On the other hand, nocodazole-induced microtubule depolymerization reduced both HCV RNA synthesis (Fig. ​(Fig.2B)2B) (9, 28) and virus release (Fig. 2A and E). Since nocodazole also blocks the movement of endosomes between pericentriolar regions and the cell periphery (3, 7), it therefore should perturb particle release. Our present results show that nocodazole had a greater effect on the reduction in extracellular HCV RNA levels (to 32%) than intracellular HCV RNA levels (to 64%) (Fig. 2A and B), and this effect may be due to blocking of endosome movement and reduced HCV RNA replication. These results can be explained on the premise that nocodazole treatments may affect both HCV RNA replication and virus release; it led us to conclude that microtubules may simultaneously play a key role in both HCV RNA replication and virus egress. Thus, both microtubules and the movement of endosomes are required for HCV particle egress.Earlier reports indicated that Rab5, an early endosomal protein, interacts with NS4B (31) and is required for HCV RNA replication. This effect was demonstrated in Rab5 small interfering RNA (siRNA)-transfected replicon cells (5, 31). However, in our current studies (Fig. ​(Fig.2B)2B) treatments with endosome inhibitors did not reduce the levels of intracellular HCV RNA. In order to investigate the discrepancy between our results and the earlier studies, we examined the effects of endosome inhibitors on the colocalization of NS5A with Rab5a in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of colocalization in the cells. Colocalization scatter diagrams were generated using the colocalization function of the Zeiss LSM Zen software. The weighted colocalization coefficient, defined as the sum of intensities of colocalizing pixels for NS5A with Rab5a in comparison to the overall sum of pixel intensities (above threshold) for NS5A, was determined. Under control conditions (DMSO), NS5A was colocalized with Rab5a throughout the cytoplasm and perinuclear region (Fig. ​(Fig.2G,2G, upper left panel). The proportion of NS5A that colocalized with Rab5a was 41% (Fig. ​(Fig.2H).2H). After treatment with U18666A, the dispersed Rab5a compartments were found only in the perinuclear region (Fig. ​(Fig.2G,2G, upper right panel). Importantly, the colocalization of NS5A with Rab5a was increased to 57% by U18666A treatment. Treatments with Baf-A1 and wortmannin, however, had no significant effect. In contrast, nocodazole treatment reduced the colocalization of NS5A with Rab5a to 12% (Fig. ​(Fig.2G2G and ​and2H).2H). Previous studies have reported that the expression levels of Rab5 and its colocalization with HCV NS4B (or NS5A) play a functional role in HCV RNA replication (31). In addition, Rab5 remained in an early endosome fraction and the expression levels of Rab5 showed no significant difference between wortmannin (100 nM)-treated or untreated cells (22). More importantly, the total levels of early endosome proteins, including EEA1 (Fig. ​(Fig.3F)3F) and Rab5a (data not shown), are not altered by the endosome inhibitors. The colocalization efficiency of NS5A with Rab5a was not reduced by U18666A, Baf-A1, or wortmannin treatments, suggesting that these drugs cannot decrease HCV RNA replication. This finding is consistent with the previous results (Fig. ​(Fig.2B).2B). These findings suggest that the observed discrepancy between our results in Fig. ​Fig.2B2B and those of the other studies (5, 31) is most likely due to differences in the expression levels of Rab5a.Open in a separate windowFIG. 3.HCV particles formed are transported from early to late endosomes. HCV-infected cells (at day 10 p.i.) were treated with the various drugs and 14 h later labeled with antibodies specific for core protein (red) and CD63 (green) (A) or EEA1 (green) (D). At the right is an enlarged area from the merged image. Nuclei were stained with DAPI (blue). (B) (E) Colocalization efficiency between core protein and early or late endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. In parallel, the cell lysates were collected and then immunoblotted with antibodies against CD63 (C) and EEA1 (F). Results were quantified by PhosphorImager counting. The HCV-infected cells (at day 10 p.i.) were fixed either for immunofluorescence microscopy (G) or for thin-section electron microscopy (H and I). Cells were costained with anti-Rab5a (green) (G, left panel), -CD63 (green) (G, right panel) and -core protein (red). Lipid droplets (LDs) and nuclei were stained with BODYPI 493/503 (blue) and DAPI (white), respectively (G). Enlarged views of parts of every image are shown (insets). (H, left panel) Early endosome (EE) (white arrow) containing particles resembling HCV adjacent to the LDs. (I, left panel) MVB/late endosome (LE) containing particles resembling HCV and internal vesicles (white arrow). High-magnification images of the early endosome (H, right panel) and MVB/late endosome (I, right panel) harboring particles resembling HCV (black arrow).To further confirm that U18666A suppresses HCV release and/or virus assembly, we determined the titer of the accumulated infectious virus particles inside the cells. The HCV-infected cells (at day 10 p.i.) were treated with U18666A at various concentrations between 2.5 and 10 μg/ml for 20 h, and then intracellular HCV particles were isolated from the cells by repeated freezing and thawing (15). The infectivity was assayed on naïve Huh7.5 cells. The results showed that the titers of infectious intracellular HCV particles were increased in a dose-dependent manner by the U18666A treatments (Fig. ​(Fig.2I).2I). These data, combined with our previous results (Fig. 2A, B, D, and E), indicate that U18666A blocks HCV particle release. Further, we determined the 50% effective dose (ED₅₀) and 50% cytotoxic concentration (CC₅₀), which are defined as the concentration of U18666A that reduced the levels of extracellular HCV RNA by 50% and the concentration of U18666A that produced 50% cytotoxicity in an MTS assay, respectively. We observed that the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h showed a dose-dependent reduction in extracellular HCV RNA levels (Fig. ​(Fig.2J).2J). The ED₅₀ and CC₅₀ were calculated by polynomial regression analysis. An ED₅₀ of 8.18 μg/ml (19.2 μM) and a CC₅₀ of 40.26 μg/ml (94.9 μM) were observed for U18666A for the reduction of extracellular HCV RNA and the cytotoxicity of HCV-infected cells, respectively. These results indicate that U18666A acts as a specific inhibitor of HCV release.In our previous results in Fig. 2A and D, we used a multiple-cycle virus growth assay, which could not discriminate the role of endosome movement in infection from that in virus assembly or egress. We therefore used a single-cycle HCV growth assay to further confirm that these endosome inhibitors could suppress HCV release. Previous studies have suggested that one round of HCV replication requires about 24 h (11). Therefore, Huh7.5 cells were infected with HCV JC1 at a multiplicity of infection (MOI) of 1 for 3 h. The HCV-infected cells were washed with phosphate-buffered saline (PBS) and then incubated with Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM). Therefore, the experiment will focus on virus egress and RNA replication instead of virus entry because the cells were first inoculated with HCV and then treated with the various drugs. At 24 h p.i., the cells and their culture supernatants were collected. Intracellular RNA and viral RNA were isolated from cell lysates and cell culture supernatants, respectively. The percentage of HCV RNA remaining after the inhibitor treatments was determined in the same way as for Fig. 2A and B. As indicated in Fig. ​Fig.2K,2K, the levels of extracellular HCV RNA (or viral particles) were reduced to 61%, 48.8%, 59.7%, and 60.4% after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively, compared to the control DMSO treatment. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 69% by nocodazole treatment (Fig. ​(Fig.2K),2K), but not by U18666A, Baf-A1, and wortmannin. Overall, these results of single-cycle HCV growth assay are similar to those of multiple-cycle virus growth assay (Fig. 2A and B), again suggesting that the endosome movement inhibitors reduced the secretion of HCV particles.To further understand the roles of the early and late endosomes in the HCV life cycle, we next examined the effects of endosome inhibitors on the colocalization of core protein with CD63 or EEA1 in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of their colocalization in the cells. The percentage of colocalization of core protein with CD63 or EEA1 was determined in the same way as for Fig. ​Fig.2H.2H. Under control conditions (DMSO), core protein was colocalized with CD63 throughout the cytoplasm, perinuclear region, and cell periphery (Fig. ​(Fig.3A,3A, top row). The proportion of core protein that colocalized with CD63 was 17% (Fig. ​(Fig.3B).3B). After treatment with U18666A, a characteristic collapse of dispersed CD63 compartments to the perinuclear region of the cells was revealed (Fig. ​(Fig.3A,3A, second row). Importantly, the colocalization of core protein with CD63 increased to 30% when the movement of late endosome was arrested by U18666A, whereas it was reduced to 2% and 7% by Baf-A1 and wortmannin treatments (Fig. 3A and B), respectively, suggesting that the movement of core protein was blocked by U18666A and accumulated in the juxtanuclear region. Additionally, EEA1-labeled distinct puncta, which are dispersed throughout the cytoplasm and partially colocalized with core protein in DMSO treatment (Fig. ​(Fig.3D,3D, top row), were found clustered in the perinuclear region following treatments with U18666A and Baf-A1 (Fig. ​(Fig.3D,3D, second and third rows). Correspondingly, the colocalization of core protein and EEA1 was increased after these treatments. In contrast, very little colocalization between core protein and EEA1 was seen after treatment with wortmannin (Fig. ​(Fig.3D,3D, bottom row). The colocalization of core protein and EEA1 was 10% in the DMSO control, in contrast with 35% and 20% after treatments with U18666A and Baf-A1, respectively, and 4% after treatment with wortmannin (Fig. ​(Fig.3E).3E). These data suggest that core protein was blocked by U18666A and accumulated in the juxtanuclear region. In parallel, the levels of CD63 and EEA1 protein in the lysates were determined by Western blotting. The results indicated that the total levels of CD63 and EEA1 were not altered by the various drug treatments (Fig. 3C and F, respectively). These results collectively indicate that the colocalization of core protein with CD63 or EEA1 and the release of virus particles depend on the motility of endosomes. Thus, we suggest that HCV core and/or the viral particles formed are transported from early to late endosomes.The above results prompted us to characterize the location of the early and late endosomes in relation to the site of core-LD colocalization, where HCV assembly takes place (23). In HCV-infected cells (at day 10 p.i.), early endosomes (Fig. ​(Fig.3G,3G, left panel) were colocalized with core protein and were located in juxtaposition to LDs, whereas the late endosomes were located far away from LDs (Fig. ​(Fig.3G,3G, right panel). These data suggest that following the assembly of viral particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes. To gain further insight into the trafficking patterns of HCV particles in Huh7.5 cells, we performed electron microscopy of the HCV-infected cells (at day 10 p.i.). Particles resembling HCV were present in both the early endosomes adjacent to the LDs (Fig. ​(Fig.3H)3H) and MVBs (late endosomes) (Fig. ​(Fig.3I).3I). The morphology of these endosomal compartments is similar to that reported previously (34, 36). These results again suggest that the HCV particles formed are transported from early to late endosomes.In order to rule out the possibility that the reduction in HCV particle secretion by endosome inhibitors (Fig. ​(Fig.2)2) was caused by the inhibition of endocytosis-mediated virus entry, we determined the percentage of cells that could be infected by HCV in the presence of endosome inhibitors. Cells were first treated with inhibitors of endosome movement, followed by HCV inoculation for 3 days. This analysis revealed that the same percentage of cells was infected and produced core protein following DMSO and U18666A treatments, 52% and 54%, respectively (Fig. 4A and B), demonstrating that U18666A did not affect HCV entry, and HCV could proceed normally to RNA replication. In contrast, Baf-A1 and wortmannin treatments yielded 7% and 20% (Fig. 4A and B), respectively, of infected cells, indicating that they blocked HCV entry, as previously reported (6, 21). Overall, these findings indicate that late endosome motility is dispensable for HCV entry and subsequent RNA replication and translation but is required for viral egress.Open in a separate windowFIG. 4.HCV entry and RNA replication are not affected by inhibiting late endosome movement, and endosomal localization of core protein is not affected by inhibiting endocytosis. Huh7.5 cells were treated with the various drugs and 14 h later washed and inoculated with HCV Jc1. At 3 days p.i., cells were stained with anti-core protein antibody (green) and DAPI (blue) (A). The images were analyzed by using Metamorph and the proportion of cells (of 5,000 counted) expressing core protein was counted (B). (C) Colocalization of HCV core protein and late endosomes is not affected by DN mutants of Esp15 or Rab5a. HCV-infected cells (at day 5 p.i.) were transfected with a control plasmid pCMV-IE (C, left panel) or with dominant negative mutants of Eps15 (pEGFP-Eps15-DN) (C, middle panel) or Rab5a (pEGFP-Rab5a-DN) (C, right panel). At day 2 posttransfection, cells were labeled with antibodies specific for core protein (blue) and CD63 (red). Cells expressed EGFP-Eps15-DN or -Rab5-DN proteins (green). Nuclei were stained with DAPI (white). Enlarged views of parts of every image (insets) are shown. Colocalization of core protein and CD63 is depicted in magenta. (D) Results from colocalization analysis are shown using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments.Moreover, we observed an almost complete block in virus infection after expression of either an Eps15 dominant negative (DN) mutant, EGFP-Eps15D95/295 (EGFP-Eps15-DN) (4), or a Rab5a dominant negative mutant, EGFP-Rab5a-S34N (EGFP-Rab5a-DN) (30), in naïve Huh7.5 cells (data not shown). This finding is consistent with a previous report (21). We further studied the effects of the DN mutants of Eps15 and Rab5a on the colocalization of core protein and the late endosomes to confirm that this colocalization was not due to the process of virus entry. Since the DN mutants of endocytosis will block HCV infection, we first performed virus infection followed by expression of the DN mutants. This strategy has been used to demonstrate that the trafficking of HIV-1 RNA and Gag protein to late endosome is independent of the endocytosed virus (19). EGFP-Eps15-DN and EGFP-Rab5-DN were transfected into HCV-infected cells (at day 5 p.i.). As shown in Fig. ​Fig.4C,4C, the localization of core protein with CD63 in these cells was not affected by the expression of these DN mutants; core protein remained well colocalized with CD63 in both the cell periphery and juxtanuclear positions in the multiple-cycle HCV growth assays. The calculated efficiency of colocalization of core protein with CD63 (19 to ∼20%; Fig. ​Fig.4D)4D) was not altered. These results indicate that core protein localization with late endosomes is not the result of the accumulation of endocytosed viruses but rather represents the trafficking intermediates of the core protein during the late viral replication stages. Thus, we conclude that the late endosome-based secretory pathways are not involved in virus entry but rather deliver the assembled virions to the extracellular milieu.Taken together, our results indicate that HCV egress requires the motility of early to late endosomes, which is microtubule dependent, and that this pathway is independent of the one required for virus entry. Thus, we postulate that following the assembly of virus particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes to the plasma membrane, where the membrane of late endosomes is fused with plasma membrane to release virions into the extracellular milieu. This transport appears to be important for HCV egress, but it is not clear how endosomes adapt in these processes. It is possible that HCV particles are transported into endosomes after their synthesis near LD. The endosome may facilitate transport of the virus particles to the plasma membrane or even to specialized cell surface domains, such as cell junctions. Notably, the tight junction protein claudin 1 has been reported to be required for HCV cell-to-cell transmission (33), which may help viruses to sequester away from the immune system.     

Association of Rice Gall Dwarf Virus with Microtubules Is Necessary for Viral Release from Cultured Insect Vector Cells

Vector insect cells infected with Rice gall dwarf virus, a member of the family Reoviridae, contained the virus-associated microtubules adjacent to the viroplasms, as revealed by transmission electron, electron tomographic, and confocal microscopy. The viroplasms, putative sites of viral replication, contained the nonstructural viral proteins Pns7 and Pns12, as well as core protein P5, of the virus. Microtubule-depolymerizing drugs suppressed the association of viral particles with microtubules and prevented the release of viruses from cells without significantly affecting viral multiplication. Thus, microtubules appear to mediate viral transport within and release of viruses from infected vector cells.Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV), and Wound tumor virus, members of the genus Phytoreovirus in the family Reoviridae, multiply both in plants and in invertebrate insect vectors. Each virus exists as icosahedral particles of approximately 65 to 70 nm in diameter, with two concentric layers (shells) of proteins that enclose a core (1, 13). The viral genome of RGDV consists of 12 segmented double-stranded RNAs that encode six structural (P1, P2, P3, P5, P6, and P8) and six nonstructural (Pns4, Pns7, Pns9, Pns10, Pns11, and Pns12) proteins (reference 21 and references therein). The core capsid is composed of P3, the major protein, which encloses P1, P5, and P6 (12). The outer layer consists of two proteins, namely, P2 and P8 (10, 12).Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of viruses in the family Reoviridae. After infecting insect vector cell monolayers (VCMs) in culture with RDV, Wei et al. (19) examined the generation of RDV particles in and at the periphery of such viroplasms. VCMs are also useful for studies of RGDV, allowing detailed analysis of the synchronous replication and multiplication of this virus (14). In order to identify the viroplasms in RGDV-infected VCMs, we examined the subcellular localization of Pns7, Pns12, P5, and RGDV particles by confocal immunofluorescence microscopy. Pns7 and Pns12 of RGDV correspond to Pns6 and Pns11, respectively, which are components of the viroplasm of RDV (12, 19). RGDV P5 is a counterpart of RDV P5, a core protein that locates inside the viroplasm in RDV-infected cells. We inoculated VCMs with RGDV, purified by the method reported in reference 15, at a multiplicity of infection (MOI) of 1; fixed them 48 h postinfection (p.i.); probed the cells with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (11, 12) that had been conjugated to fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO) or rhodamine (Sigma); and examined them by confocal microscopy, as described previously (19). In RGDV-infected cells, Pns7, Pns12, and P5 were detected as punctate inclusions (Fig. ​(Fig.1).1). Immunostained viral antigens formed ringlike structures around the punctate inclusions. When the images were merged, Pns7, Pns12, and P5 were colocalized in the punctate inclusions, indicating that these proteins were constituents of the viral inclusions (Fig. ​(Fig.1).1). Our observations revealed the similar respective localizations of the corresponding nonstructural proteins, core proteins, and viral particles of two phytoreoviruses, RGDV and RDV, in infected cells. Thus, Pns7 and Pns12 of RGDV had attributes common to their functional counterparts—Pns6 and Pns11, respectively—of RDV (19). The core protein P5 was located inside the viroplasms, and the viral antigens were distributed at the periphery of the viroplasms. The results, together, suggest that RGDV and RDV exploit similar replication strategies. Specific fluorescence was not detected in noninfected cells after incubation with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (data not shown).Open in a separate windowFIG. 1.Subcellular localization of Pns7, Pns12, and P5 of RGDV and viral antigens in RGDV-infected VCMs 48 h p.i. Arrowheads show ringlike profiles of viral antigens that surround viral inclusions, which have been immunostained with the Pns12-specific antibodies. Arrows show the fibrillar profiles of immunostained viral antigens. Bars, 5 μm.In addition to the viral location at the periphery of the viral inclusions visualized as immunostained Pns12 (Fig. ​(Fig.1),1), the antigens were distributed as bundles of fibrillar structures, a form not observed in RDV-infected cells. To analyze the entity of the bundles of fibrillar structures, VCMs on coverslips were inoculated with RGDV at an MOI of 1, fixed at 48 h p.i., and examined by electron microscopy (EM), as described previously (19). We observed viral particles of approximately 70 nm in diameter in close association with the free ends, as well as along the edges, of tubules of approximately 25 nm in diameter (Fig. 2A to D). The abundant bundles of tubules with closely associated viral particles were clearly in contact with the periphery of granular, electron-dense inclusions of 800 to 1,200 nm in diameter (Fig. ​(Fig.2B),2B), namely, viroplasms. The dimensions and appearance of the tubular structures resembled those of microtubules (Fig. ​(Fig.2C)2C) (17). Transverse sections of tubules revealed arrays of closed circles of approximately 25 nm in diameter, with viral particles attached directly or via a filament to the circumference (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.Association of RGDV particles with microtubules. (A) Electron micrograph showing RGDV particles associated with microtubules in virus-infected VCMs 48 h p.i. Bar, 300 nm. VP, electron-dense inclusion. (B) Virus-associated microtubules in contact with the periphery of the electron-dense inclusion indicated by a white rectangle in panel A. Bar, 300 nm. (C) Viral particles along the edges of tubules of approximately 25 nm in diameter. Bar, 150 nm. (D) Transverse sections of arrays of closed circles of approximately 25 nm in diameter with viral particles attached to their circumference directly (arrow) or via a filament (arrowhead). Bar, 150 nm. (E) Confocal micrograph showing the association of viral particles with microtubules in virus-infected VCMs 48 h p.i. Microtubules were stained with α-tubulin-specific antibodies conjugated to FITC; viral particles were stained with viral-antigen-specific antibodies conjugated to rhodamine. Arrowheads indicate the ringlike organization of viral antigens. Arrows show the colocalization of fibrillar profiles of viral antigens with microtubules. The insets show ringlike and fibrillar profiles of immunostained viral antigens. The circular areas inside the ringlike structures are viroplasms. Bar, 5 μm.Our observations suggested that RGDV particles might attach to microtubules in infected cells. To examine this possibility, we inoculated VCMs with RGDV at an MOI of 1, fixed the cells 48 h p.i., immunostained them with α-tubulin-specific antibodies conjugated to FITC and with viral-antigen-specific antibodies conjugated to rhodamine, and analyzed them by confocal microscopy, as described previously (19). Viral antigens were visualized as ringlike and fibrillar structures (Fig. ​(Fig.2E).2E). Double immunostaining of the infected cells revealed that a network of microtubule-based filaments colocalized with most of the fibrillar structures that represented viral antigens, confirming the association of viral particles with the microtubule-like inclusions visualized by EM (Fig. ​(Fig.2A).2A). Nonspecific reactions were not detected with either of the stainings (data not shown). Our results suggested that RGDV particles, which assembled at the periphery of viroplasms, might be transported along microtubules. Due to the lack of RGDV infectious clones fused with green fluorescent protein and the effective gene transfection system for VCMs, we could not observe the trafficking of RGDV particles along microtubules in living cells.We then used three-dimensional (3-D) electron tomographic microscopy (ET) to reveal a new level of morphological detail about the association of RGDV with microtubules. To produce 3-D reconstructions of RGDV-infected cells, we fixed, embedded, and sectioned infected leafhopper cells as described previously (5). We chose a representative region that showed numerous RGDV particles close to bundles of microtubules for this novel tomographic analysis. A single-axis tilt series was collected manually from −60° to 60° with 2° increments using an H9500SD EM (Hitachi, Tokyo) operated at 200 kV. These tomographic data were recorded at a defocus of 3.6 μm on the TVIPS 2k × 2k charge-coupled-device camera (TVIPS, Gauting, Germany). Microscopic magnification of ×15,000, providing 1.28 nm/pixel, was enough to view the microtubules and virus particles following tomographic reconstruction of the tilt series using IMOD (7). As shown in the 3-D tomogram in Fig. ​Fig.3,3, most of the RGDV particles were bound to the edges of bundles of microtubules. The RGDV particles along the edges of microtubules were arrayed in an orderly but uncrowded manner (Fig. ​(Fig.3).3). Our ET analysis also revealed that some viral particles were linked to filaments of approximately 10 nm in diameter (Fig. ​(Fig.3B).3B). Morphologically, these filaments resembled vimentin intermediate filaments (4). In many lines of cultured cells, vimentin intermediate filaments partially overlap the microtubules, and there is evidence that the two filament systems interact (3, 9, 20). Unfortunately, vimentin-specific monoclonal antibodies from mouse and rabbit did not react specifically with our leafhopper cells (data not shown), but the nature of the intermediate filaments was apparent from their dimensions, intracellular location, and organization. Thus, our ET analysis indicated that RGDV particles were able to associate directly and/or via intermediate filaments with microtubules.Open in a separate windowFIG. 3.ET analysis showing the association of RGDV particles with microtubules either directly or via intermediate filaments. (A) Translucent representation of the reconstructed viruses lining up with microtubules. (B) Slice of the reconstructed volumes from the inset of A to show the association of RGDV particles with intermediate filaments (arrows). Bars, 150 nm.To examine the role of the microtubules for RGDV activity, we added a microtubule-disrupting agent, either nocodazole (Sigma) or colchicine (Sigma), 2 hours after inoculation of VCMs with RGDV at an MOI of 1 and then continued the incubation for a further 46 h. Cells were fixed 48 h p.i. and stained with α-tubulin-specific antibodies conjugated to FITC (Sigma) and viral-particle-specific antibodies conjugated to rhodamine, with subsequent confocal fluorescence microscopy, as described previously (19). We tested a range of drug concentrations in preliminary experiments (data not shown) and determined optimal concentrations. Treatment of infected cells with 10 μM nocodazole or 5 μg/ml colchicine resulted in the complete disassembly of microtubules, with the accumulation of ringlike structures exclusively and no fibrillar structures representative of viral antigens in the cytoplasm (Fig. ​(Fig.4A).4A). These ringlike aggregates of viral antigens were confirmed to surround viroplasms when the latter were immunostained for Pns12, as described above and shown in Fig. ​Fig.1.1. Nonspecific reactions were not detected with either staining (data not shown). These results suggest that RGDV particles multiply around the viroplasm but are unable to distribute along the microtubules in the presence of the chemicals.Open in a separate windowFIG. 4.(A) Effects of microtubule-disrupting agents on the formation of microtubules and fibrillar profiles of immunostained viral antigens. Bars, 5 μm. The insets show the ringlike profiles of immunostained viral antigens after treatment with inhibitors, suggesting that viral replication occurs in the presence of each inhibitor. (B) Effects of drugs on the production of cell-associated (gray bars) and extracellular (black bars) viruses in VCMs infected with RGDV. The error bars indicate standard deviations.During the process of infection, microtubules play important roles in viral entry, intracellular trafficking, and extracellular release (2, 8, 16). We next investigated the effects of the microtubule-disrupting agents on the production in and release of viruses from virus-infected cells by the method described previously (18). Nocodazole or colchicine was added 2 h after inoculation of VCMs with RGDV at an MOI of 1, and incubation was continued for a further 46 h. The extracellular medium and the cells were collected separately. The medium was centrifuged for 30 min at 15,000 × g, and the supernatant was collected. The cells were subjected to three cycles of freezing and thawing to release viral particles. The viral titer of each sample was determined, in duplicate, by the fluorescent focus assay as described previously (6), with VCMs and a magnification of ×10. As shown in Fig. ​Fig.4B,4B, nocodazole (20 μM) and colchicine (10 μg/ml) caused a fivefold reduction in the number of released viruses, compared to that from untreated control infected cells. In contrast, each inhibitor at the selected dose failed to significantly reduce the titer of cell-associated viruses (less then 5% compared to that from untreated control). These results suggest that the inhibitors impeded the release of viruses into the medium without affecting viral production in infected cells. We do not yet understand why the viral titer was not elevated in drug-treated cells from which viral release was inhibited. However, our data show clearly that disruption of microtubules directly inhibited the release of mature viral particles from infected cells.In conclusion, EM, ET, immunofluorescence staining, and experiments with two inhibitors support the hypothesis that the transport of RGDV from viroplasms to the plasma membrane and into the medium is dependent on microtubules. In the case of RDV, vesicular compartment-containing viral particles that locate adjacent to the viroplasms were considered to play an important role in the transport and release of the virus from the viroplasm to the culture medium in infected VCMs (18). On the other hand, a fibrillar structure (Fig. ​(Fig.11 and ​and2),2), not observed in RDV-infected cells, was considered to function in the trafficking of RGDV from viroplasm into the culture medium (Fig. ​(Fig.4)4) in the present study. RGDV and RDV, both members of the Phytoreovirus genus, have some common biological and biochemical properties but are distinct from each other (13). For example, viruses are restricted to phloem-related cells in RGDV-infected plants but distributed in many types of cells in RDV-infected plants, and a P2 protein with a function to adsorb to and/or penetrate into insect vector cells is present in RGDV and absent in RDV in particles purified using carbon tetrachloride. The present molecular cytopathological study revealed one more difference between the viruses: they have different means for transporting and releasing infectious particles to the cell exterior. The presence of such a molecular mechanism may accelerate the secondary infections by the viruses in infected vector insects, and the high propagation speed would allow the viruses to complete infection cycles through insects and plants.     

Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.We have previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway (15) to higher alcohols (3). In order to produce isobutanol, the valine biosynthesis pathway was used to generate 2-ketoisovalerate (KIV), the precursor to valine, which was then converted to isobutanol via a decarboxylation and reduction step (Fig. ​(Fig.1A).1A). The entire pathway to isobutanol from glucose is shown in Fig. ​Fig.1A.1A. To produce isobutanol, we overexpressed five genes, alsS (Bacillus subtilis), ilvC (Escherichia coli), ilvD (E. coli), kdc (Lactococcus lactis), and ADH2 (Saccharomyces cerevisiae) (Fig. ​(Fig.1A).1A). This E. coli strain produced 6.8 g/liter isobutanol in 24 h (Fig. ​(Fig.1B)1B) and more than 20 g/liter in 112 h (3). More recently, we have found that an alcohol dehydrogenase (Adh) encoded by yqhD on the E. coli genome can convert isobutyraldehyde to isobutanol efficiently (5) (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Schematic representation of the pathway for isobutanol production. (A) The Kdc-dependent synthetic pathway for isobutanol production. (B) Isobutanol production with the Kdc-dependent and -independent synthetic pathways. IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase. (C) Enzymatic reaction of Als, Ahbs, and Kdc activities.One key reaction in the production of isobutanol is the conversion of KIV to isobutyraldehyde catalyzed by 2-ketoacid decarboxylase (Kdc) (Fig. ​(Fig.1C).1C). Since E. coli does not have Kdc, kdc from L. lactis was overexpressed. Kdc is a nonoxidative thiamine PP_i (TPP)-dependent enzyme and is relatively rare in bacteria, being more frequently found in plants, yeasts, and fungi (8, 19). Several enzymes with Kdc activity have been found, including pyruvate decarboxylase, phenylpyruvate decarboxylase (18), branched-chain Kdc (8, 19), 2-ketoglutarate decarboxylase (10, 17, 20), and indole-3-pyruvate decarboxylase (13).In this work, unexpectedly, we find that Kdc is nonessential for E. coli to produce isobutanol (Fig. ​(Fig.1).1). An E. coli strain overexpressing only alsS (from B. subtilis), ilvC, and ilvD (both from E. coli) is still able to produce isobutanol. Since E. coli is not a natural producer of isobutanol, it cannot be detected from the culture media in any unmodified strain. We identify that AlsS from B. subtilis, which was introduced in E. coli for acetolactate synthesis (Als), catalyzes the decarboxylation of 2-ketoisovalerate like Kdc both in vivo and in vitro. AlsS is part of the acetoin synthesis pathway and catalyzes the aldo condensation of two molecules of pyruvate to 2-acetolactate (Als activity) (Fig. ​(Fig.1C)1C) (11). The overall reaction catalyzed by AlsS is irreversible because of CO₂ evolution. The first step in catalysis is the ionized thiazolium ring of TPP reacting with the first pyruvate, followed by decarboxylation. This intermediate then reacts with the second pyruvate. Deprotonation followed by C-C bond breakage produces 2-acetolactate. In this work, mutational approaches were used to assess the importance of Q487 in the Kdc activity of AlsS.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.