Proteasome Inhibition In Vivo Promotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.Severe acute respiratory syndrome (SARS) was first introduced into the human population in the Guangdong Province in China and rapidly spread to several other countries (31). SARS is caused by infection with the SARS coronavirus (SARS-CoV) and is characterized by an atypical pneumonia and lymphopenia. Two-thirds of the SARS-infected patients developed a viral pneumonitis, of which 10% developed acute respiratory distress syndrome. During the outbreak in 2002 to 2003, 8,000 people were infected and 774 people died from respiratory failure (36; WHO, Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 [http://www.who.int]). At present there are no effective treatments for SARS as well as other coronavirus infections. Finding an effective treatment for coronavirus infections could be protective in the event of a reemergent coronavirus outbreak (7).We have recently reported that a rodent model of SARS mimics many of the features of severe clinical SARS pathology (11, 12). Intranasal infection of A/J mice with strain 1 of murine hepatitis virus (MHV-1) causes a lethal form of pneumonitis, characterized by marked innate immune inflammatory cytokine production and replication of the virus in pulmonary macrophages (11, 12). MHV-1 infection is uniformly fatal in infected A/J mice; the resultant disease serves as a model to understand the pathology of the most severe SARS cases. In mice, the pulmonary damage is histologically similar to that seen in human SARS and is similarly associated with a marked upregulation of inflammatory mediators, including monocyte chemoattractant protein 1 (MCP-1), IP-10, MIG, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 12, 25). These innate immune mediators are likely to play roles in human SARS and MHV-1 SARS-like pathogenesis.A critical aspect of the host innate immune response to viral illness is the upregulation of the antiviral type 1 IFN response. With respect to SARS, type 1 IFN responses have been reported to be suppressed by SARS-CoV in several models and in clinical cases (11, 39, 45, 52). In our model, MHV-1-infected A/J mice produce less type 1 IFN than resistant strains of mice and they respond poorly to IFN-β therapy (11). Type I IFN has been used clinically in the treatment of established SARS infections but has shown only limited efficacy (25). In the absence of an effective antiviral treatment, the innate immune pathways present a potential target for therapeutic intervention (7).Ubiquitination, the process by which cellular proteins are conjugated to the 7.5-kDa ubiquitin (Ub) protein, is a critical regulator of innate and adaptive immune pathways (40). There are several possible fates for ubiquitinated proteins: degradation by the 26S proteasome, trafficking to various subcellular sites, altered interactions with other proteins, and altered signal transduction functions (28). The fates of the ubiquitinated proteins, many of which overlap, can play a role in innate immunity. Since the first discovery that papillomavirus encodes an E3 ubiquitin ligase that targets p53, it has become widely appreciated that many viruses encode proteins that target or exploit ubiquitination pathways (37, 43). For example, Epstein-Barr virus and herpes simplex virus proteins interact with the host deubiquitinating (DUB) protein USP7 (13, 17). Ubiquitination of IRF3 has been implicated in the viral control of the innate immune system (22, 48, 49). DUB may also be important for viral functions, such as the assembly of viral replicase proteins with double-membrane vesicles at the site of replication, a process that parasitizes autophagy (39).All coronaviruses, including MHV (A59 and JHM), infectious bronchitis virus, and human CoV229E SARS coronavirus, encode one or more papain-like proteases (PLpros) (PL1pro and PL2pro) (3, 5, 19, 23, 50). One role for the PL2pro proteases is to cleave the coronavirus polyprotein into its component parts. This enzyme, isolated from the SARS-CoV, has also been shown to have DUB activity both in vitro and in HeLa cells (23), suggesting that it might also play a role in modulating the host ubiquitination pathways. PLpro proteases harbor an N-terminal Ub-like domain reported to mediate interactions between PLpro DUB activity and the cellular proteasome (35). Although there is no direct link between the proteasome and SARS-CoV DUB activity, the presence of the Ub1 domain and of SARS-CoV DUB activity suggests that the proteasome may be being exploited by the virus either to evade the immune response or to promote viral replication. These interactions also suggest that the ubiquitination system might be a target for antiviral therapeutic intervention.We explored the role of the cellular proteasome in MHV-1 replication and in the innate immune response to the virus by testing the effects of small-molecule proteasome inhibitors in both cell-based and murine models of SARS pneumonitis. We compared the results in the SARS model to a well-described model of lymphocytic choriomeningitis virus (LCMV) hepatitis in order to test for virus-specific effects. To control for nonspecific effects of the inhibitors, we used three different agents: pyrrolidine dithiocarbamate (PDTC), MG132, and PS-341 (bortezomib, Velcade). PDTC is a chelating agent that reversibly inhibits the proteasome complex, MG132 is a peptide aldehyde protease inhibitor, and PS-341 is a peptide boronic acid inhibitor (1, 20, 38). PS-341 is a clinically approved drug currently being used in the treatment of multiple myeloma.     

Toll-Like Receptor 4 Deficiency Increases Disease and Mortality after Mouse Hepatitis Virus Type 1 Infection of Susceptible C3H Mice

Severe acute respiratory syndrome (SARS) is characterized by substantial acute pulmonary inflammation with a high mortality rate. Despite the identification of SARS coronavirus (SARS-CoV) as the etiologic agent of SARS, a thorough understanding of the underlying disease pathogenesis has been hampered by the lack of a suitable animal model that recapitulates the human disease. Intranasal (i.n.) infection of A/J mice with the CoV mouse hepatitis virus strain 1 (MHV-1) induces an acute respiratory disease with a high lethality rate that shares several pathological similarities with SARS-CoV infection in humans. In this study, we examined virus replication and the character of pulmonary inflammation induced by MHV-1 infection in susceptible (A/J, C3H/HeJ, and BALB/c) and resistant (C57BL/6) strains of mice. Virus replication and distribution did not correlate with the relative susceptibilities of A/J, BALB/c, C3H/HeJ, and C57BL/6 mice. In order to further define the role of the host genetic background in influencing susceptibility to MHV-1-induced disease, we examined 14 different inbred mouse strains. BALB.B and BALB/c mice exhibited MHV-1-induced weight loss, whereas all other strains of H-2^b and H-2^d mice did not show any signs of disease following MHV-1 infection. H-2^k mice demonstrated moderate susceptibility, with C3H/HeJ mice exhibiting the most severe disease. C3H/HeJ mice harbor a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts TLR4 signaling. C3H/HeJ mice exhibit enhanced morbidity and mortality following i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.Severe acute respiratory syndrome (SARS) is a disease that was initially observed in 2002 and led to approximately 8,000 affected individuals in multiple countries with over 700 deaths (1, 24, 47, 48). The causative agent of SARS was subsequently identified as a novel coronavirus (CoV) termed SARS-CoV (8, 17, 22, 27, 32, 37). Although SARS-CoV infections following the initial outbreak in 2002 and 2003 have been limited primarily to laboratory personnel, the identification of an animal reservoir for the virus raises concern about the potential for future outbreaks (25).The pathogenesis of SARS has been difficult to study, in part because no animal model is able to fully recapitulate the morbidity and mortality observed in infected humans (35). Infection of a number of inbred mouse strains, including BALB/c, C57BL/6, and 129S, with primary human isolates of SARS-CoV results in the replication of the virus within the lung tissue without the subsequent development of readily apparent clinical disease (11, 16, 41). Infection of aged BALB/c mice results in clinically apparent disease that more closely mimics some aspects of SARS in humans (36). However, immune responses in aged mice are known to be altered (5, 15), and thus, the mechanisms that control the induction of disease may differ between adult and aged mice. Recent work has demonstrated that serial passage of SARS-CoV in mice results in a mouse adaptation that leads to more profound replication of the virus in the lung (28, 34). However, the time to death from this mouse-adapted SARS-CoV is 3 to 5 days, which is much more rapid than the time to mortality observed in fatal cases of SARS in humans.Phylogenetic analysis has revealed that SARS-CoV is most closely related to group 2 CoVs, which include the mouse hepatitis virus (MHV) family (39). Thus, information gathered by infection of mice with closely related members of the group 2 CoVs may further contribute to our understanding of SARS-CoV pathogenesis in humans. While many strains of MHV induce primarily hepatic and central nervous system diseases (6, 7, 12, 18, 21, 23, 40), a recent study demonstrated that intranasal (i.n.) infection of A/J mice with MHV type 1 (MHV-1) induces pulmonary injury that shares several pathological characteristics with SARS-CoV infection of humans (2, 3, 9, 29, 43).In the current study, we examined the relationship between MHV-1 replication in the lungs and the severity of disease in four inbred strains of mice: A/J, BALB/c, C57BL/6, and C3H/HeJ. Our results demonstrate that MHV-1 replicates to similar levels in the lung in each of these inbred strains of mice regardless of their relative levels of susceptibility, as measured by weight loss and clinical illness. Both A/J and C3H/HeJ mice exhibited enhanced weight loss and clinical illness following i.n. MHV-1 infection compared to BALB/c and C57BL/6 mice. Analysis of many different inbred mouse strains confirmed A/J and C3H/HeJ mice as the most susceptible to i.n. MHV-1 infection. Interestingly, C3H/HeJ mice harboring a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts its normal function exhibited greatly increased morbidity and mortality after i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.     

Cellular Immune Responses to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection in Senescent BALB/c Mice: CD4+ T Cells Are Important in Control of SARS-CoV Infection

We characterized the cellular immune response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in 12- to 14-month-old BALB/c mice, a model that mimics features of the human disease. Following intranasal administration, the virus replicated in the lungs, with peak titers on day 2 postinfection. Enhanced production of cytokines (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokines (CXCL10, CCL2, CCL3, and CCL5) correlated with migration of NK cells, macrophages, and plasmacytoid dendritic cells (pDC) into the lungs. By day 7, histopathologic evidence of pneumonitis was seen in the lungs when viral clearance occurred. At this time, a second wave of enhanced production of cytokines (TNF-α, IL-6, gamma interferon [IFN-γ], IL-2, and IL-5), chemokines (CXCL9, CXCL10, CCL2, CCL3, and CCL5), and receptors (CXCR3, CCR2, and CCR5), was detected in the lungs, associated with an influx of T lymphocytes. Depletion of CD8⁺ T cells at the time of infection did not affect viral replication or clearance. However, depletion of CD4⁺ T cells resulted in an enhanced immune-mediated interstitial pneumonitis and delayed clearance of SARS-CoV from the lungs, which was associated with reduced neutralizing antibody and cytokine production and reduced pulmonary recruitment of lymphocytes. Innate defense mechanisms are able to control SARS-CoV infection in the absence of CD4⁺ and CD8⁺ T cells and antibodies. Our findings provide new insights into the pathogenesis of SARS, demonstrating the important role of CD4⁺ but not CD8⁺ T cells in primary SARS-CoV infection in this model.The global outbreak of severe acute respiratory syndrome (SARS) in 2003 that infected more than 8,000 people in 29 countries across five continents, with 774 deaths reported by the World Health Organization (54), was caused by a highly contagious coronavirus designated SARS-CoV (33). The elderly were more likely to die from SARS-CoV infection than younger people (7), with a case-fatality rate of 50% in people older than 65 years (14, 53). Disease pathogenesis in SARS is complex, with multiple factors leading to severe pulmonary injury and dissemination of the virus to other organs. High viral load; systemic infection; a cytokine storm with high levels of CXCL10/IP-10, CCL3/MIP-1α, and CCL2/MCP-1; massive lung infiltration by monocytes and macrophages; and rapid depletion of T cells are hallmarks of SARS (5, 13, 15, 21, 28, 35). The role of neutralizing antibodies (Abs) in protection from SARS-CoV infection has been well documented. Virus-specific neutralizing Abs reduce viral load, protect against weight loss, and reduce histopathology in animal models (42, 47, 48). Although the role of type I interferons (IFNs) in the natural history of SARS is controversial (5, 9, 59), the innate defense system appears to be critical for controlling SARS-CoV replication in mice (23, 41). Mice lacking normal innate signaling due to STAT1 or MyD88 deficiency are highly susceptible to SARS-CoV infection. Virus-specific T-cell responses are present in convalescent patients with SARS (27, 55). However, little is known about the role of T cells in the acute phase of SARS.Several mouse models have been developed for the in vivo study of SARS pathogenesis. However, no single model accurately reproduces all aspects of the human disease. SARS-CoV replicates in the upper and lower respiratory tracts of 4- to 8-week-old mice and is cleared rapidly; infection is associated with transient mild pneumonitis, and cytokines are not detectable in the lungs (20, 42, 49). A SARS-CoV isolate that was adapted by serial passage in mice (MA-15) replicates to a higher titer and for a longer duration in the lungs than the unadapted (Urbani) virus and is associated with viremia and mortality in young mice (36), but the histologic changes in the lungs are caused by high titers of virus and cell death without significant infiltrates of inflammatory cells. The heightened susceptibility of elderly patients to SARS led us to develop a pneumonia model in 12- to 14-month-old (mo) BALB/c mice using the Urbani virus. In this model, pulmonary replication of virus was associated with signs of clinical illness and histopathological evidence of disease characterized by bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring (3), thus resembling SARS in the elderly. We evaluated the host response to SARS-CoV infection by examining the gene expression profile in the senescent mouse model and found a robust response to virus infection, with an increased expression of several immune response and cell-to-cell signaling genes, including those for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), CCL2, CCL3, CXCL10, and IFN-γ (1).In this study, we characterize the cellular immune response to SARS-CoV infection in 12- to 14-mo BALB/c mice in terms of the protein and gene expression of inflammatory mediators, migration of inflammatory cells, and virus-specific T-cell responses in the lungs during the course of disease. We evaluated the role of T cells in disease pathogenesis and viral clearance by depleting T-cell subsets at the time of infection and found an important role for CD4⁺ T cells (but not CD8⁺ T cells) in primary infection with SARS-CoV in this model.     

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca²⁺-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8, 14, 18), where it activates the complement system or acts directly as an opsonin (30, 40, 52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (8, 14, 18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (13, 37, 67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (29, 42, 72, 73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (29, 73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (42).In this study, retroviral particles pseudotyped with SARS-S and in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.     

A Single Tyrosine in the Severe Acute Respiratory Syndrome Coronavirus Membrane Protein Cytoplasmic Tail Is Important for Efficient Interaction with Spike Protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y₁₉₅, was important for S-M interaction. When Y₁₉₅ was mutated to alanine, M_Y195A no longer retained S intracellularly at the Golgi. Unlike wild-type M, M_Y195A did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y₁₉₅ also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y₁₉₅ is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.Coronaviruses are enveloped positive-strand RNA viruses that infect a wide variety of mammalian and avian species. These viruses generally cause mild disease in humans and are one major cause of the common cold (34). However, severe acute respiratory syndrome coronavirus (SARS-CoV), a novel human coronavirus which emerged in the Guangdong province in China in 2002 (30, 48), caused a widespread pandemic. SARS-CoV caused severe disease with a mortality rate of approximately 10%, the highest for any human coronavirus to date (62). The phylogeny and group classification of SARS-CoV remain controversial (17), but it is widely accepted to be a distant member of group 2. While SARS-CoV is no longer a major health threat, understanding the basic biology of this human pathogen remains important.Coronaviruses encode three major envelope proteins in addition to various nonstructural and accessory proteins. The envelope protein (E) is the least abundant structural protein in the virion envelope, although it is expressed at robust levels during infection (21). E plays an essential role in assembly for some but not all coronaviruses (31-33, 45) and may also be a viroporin (reviewed in reference 21). The spike glycoprotein (S) is the second most abundant protein in the envelope. S determines host cell tropism, binds the host receptor, and is responsible for virus-cell, as well as cell-cell, fusion (15). The S protein is a type I membrane protein with a large, heavily glycosylated luminal domain and a short cytoplasmic tail that has been shown to be palmitoylated in some coronaviruses (47, 58). The membrane protein (M) is the most abundant protein in the virion envelope and acts as a scaffold for virus assembly. M has three transmembrane domains, a long cytoplasmic tail, and a short glycosylated luminal domain (reviewed in reference 21). Unlike many enveloped viruses, coronaviruses assemble at and bud into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and exit the infected cell by exocytosis (29). In order to accomplish this, the envelope proteins must be targeted to the budding compartment for assembly.For most coronaviruses, the E and M proteins localize in the Golgi region near the budding site independently of other viral structural proteins. We have previously shown for infectious bronchitis virus (IBV) E protein that the cytoplasmic tail contains Golgi targeting information (9). IBV M contains Golgi targeting information in its first transmembrane domain (57), while the transmembrane domains and cytoplasmic tail of mouse hepatitis virus (MHV) M appear to play a role in Golgi targeting (1, 36). Some coronavirus S proteins contain targeting information in their cytoplasmic tails; however, some do not (38, 39, 52, 63). Since S proteins can escape to the cell surface when highly expressed, S may rely on lateral interactions with other viral envelope proteins to localize to the budding site and be incorporated into newly assembling virions.In line with its role in virus assembly, M is necessary for virus-like particle (VLP) formation (3, 10, 26, 40, 55, 59). M has been shown to interact with itself to form homo-oligomers (12). In addition, M interacts with E, S, and the viral nucleocapsid and is essential for virion assembly (reviewed in reference 21). Lateral interactions between the coronavirus envelope proteins are critical for efficient virus assembly. The interaction of S and M has been studied for MHV, and the cytoplasmic tail of each protein is important for interaction (16, 44). Specifically, deletion of an amphipathic region in the MHV M cytoplasmic tail abrogates efficient interaction with MHV S (11). The S and M proteins of IBV, bovine coronavirus, feline infectious peritonitis virus, and SARS-CoV have been shown to interact; however, information about the specific regions that are important for interaction remains elusive (16, 22, 26, 42, 64). Due to the presence of several accessory proteins in the virion envelope (23-25, 28, 51, 53), it is possible that the requirements for SARS-CoV S and M interaction could be different from those of previously studied coronaviruses.In earlier work, we reported that SARS-CoV M retains SARS-CoV S intracellularly at the Golgi region when both proteins are expressed exogenously (39). We also demonstrated that the SARS-CoV S cytoplasmic tail interacts with in vitro-transcribed and -translated SARS-CoV M (39). Here, we show that the SARS-CoV M cytoplasmic tail is necessary for specific retention of SARS-CoV S at the Golgi region. We found a critical tyrosine residue at position 195 to be important for retaining SARS-CoV S Golgi membranes when coexpressed with M. When Y₁₉₅ was mutated to alanine, the mutant protein, M_Y195A, did not reduce the amount of SARS-CoV S at the plasma membrane or reduce the extent of S carbohydrate processing as well as wild-type SARS-CoV M does. Additionally, mutation of Y₁₉₅ in SARS-CoV M disrupted the S-M interaction in vitro. Thus, Y₁₉₅ is likely to play a critical role in the assembly of infectious SARS-CoV.     

T Cell Responses Are Required for Protection from Clinical Disease and for Virus Clearance in Severe Acute Respiratory Syndrome Coronavirus-Infected Mice

A dysregulated innate immune response and exuberant cytokine/chemokine expression are believed to be critical factors in the pathogenesis of severe acute respiratory syndrome (SARS), caused by a coronavirus (SARS-CoV). However, we recently showed that inefficient immune activation and a poor virus-specific T cell response underlie severe disease in SARS-CoV-infected mice. Here, we extend these results to show that virus-specific T cells, in the absence of activation of the innate immune response, were sufficient to significantly enhance survival and diminish clinical disease. We demonstrated that T cells are responsible for virus clearance, as intravenous adoptive transfer of SARS-CoV-immune splenocytes or in vitro-generated T cells to SCID or BALB/c mice enhanced survival and reduced virus titers in the lung. Enhancement of the number of virus-specific CD8 T cells by immunization with SARS-CoV peptide-pulsed dendritic cells also resulted in a robust T cell response, earlier virus clearance, and increased survival. These studies are the first to show that T cells play a crucial role in SARS-CoV clearance and that a suboptimal T cell response contributes to the pathological changes observed in SARS. They also provide a new approach to SARS vaccine design.Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in over 8,000 cases of respiratory disease, with 10% mortality, in 2002 and 2003 (18). Patients with severe disease developed acute lung injury (ALI), concomitant with neutrophilia, lymphopenia, and prolonged expression of several proinflammatory cytokines (4, 17, 18, 32). Virus was cleared slowly from these patients and could be detected in respiratory secretions for as long as 21 days postinfection (p.i.) (14, 17).SARS has not recurred to a significant extent since 2003, so most studies have used animal infections to investigate the mechanism of severe disease. These studies have been facilitated by the isolation and characterization of SARS-CoV variants that cause severe respiratory disease in mice or rats after adaptation to these hosts by serial passage (15, 16, 22). One strain, isolated after 15 passages through the lungs of BALB/c mice, caused respiratory disease in young BALB/c mice (MA15 virus) (22). As in humans with severe disease, cytokine expression is elevated and prolonged in animals with severe disease, whether caused by the human Urbani or rodent-adapted strains of SARS-CoV, and is accompanied by delayed kinetics of virus clearance (1, 5, 15, 23, 26, 35). This elevated expression of proinflammatory mediators is believed to be a major contributory factor in the development of pneumonia and, subsequently, ALI.These studies did not address the role of the antivirus T cell response in disease. T cell responses are critical for virus clearance and protection from clinical disease in mice infected with other coronaviruses, such as mouse hepatitis virus (MHV) (31), or other pulmonary pathogens, such as influenza A virus or Sendai virus (7, 29). Although some studies using the Urbani strain showed that virus clearance was T cell independent (5, 34), mice infected with this strain develop only mild pneumonitis, so that the requirement for a virus-specific T cell response for protection in SARS-CoV-infected animals remains unclear.Young BALB/c mice infected with MA15 virus develop severe respiratory disease, with substantial mortality, dependent upon virus dosage (22). We recently showed that the pulmonary immune response is inefficiently activated in these mice at early times after infection, resulting in a barely detectable antivirus T cell response in mice with a lethal infection (35). Survival correlated with the development of a SARS-CoV-specific CD4 and CD8 T cell response. Poor activation of the immune response could be reversed by depletion of inhibitory alveolar macrophages with clodronate, a drug that depletes phagocytic cells (28, 30), or by treatment with poly(I-C) or CpG, both of which activate macrophages and dendritic cells (DCs). Disease could also be ameliorated by adoptive transfer of activated DCs, which are able to traffic to the lung draining lymph nodes (DLN) and prime the antivirus T cell response. These interventions effect two changes in the host immune response: they result in activation of the innate immune system and facilitate the development of a robust virus-specific T cell response. From these results, we could not determine the relative importance of these two limbs of the immune response in protection. Here, we examined whether a robust antivirus T cell response was sufficient to protect mice in the absence of interventions that activated the innate immune response. Our results indicate that virus-specific T cells are necessary and sufficient for virus clearance and for protection from clinical disease in MA15 virus-infected mice.     

Novel Immunodominant Peptide Presentation Strategy: a Featured HLA-A*2402-Restricted Cytotoxic T-Lymphocyte Epitope Stabilized by Intrachain Hydrogen Bonds from Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β₂-microglobulin (β₂m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.In 2003, severe and acute respiratory syndrome (SARS), emerging from China, caused a global outbreak, affecting 29 countries, with over 8,000 human cases and greater than 800 deaths (5, 9, 24, 33, 37). Thanks to the unprecedented global collaboration coordinated by the WHO, SARS coronavirus (SARS-CoV), a novel member of Coronaviridae family, was rapidly confirmed to be the etiological agent for the SARS epidemic (36). Soon after the identification of the causative agent, SARS was controlled and then quickly announced to be conquered through international cooperation on epidemiological processes (9). However, the role that human immunity played in the clearance of SARS-CoV and whether the memory immunity will persist for the potential reemergence of SARS are not yet well understood.In viral infections, CD8⁺ cytotoxic T lymphocytes (CTLs) are essential to the control of infectious disease. Virus-specific CD8⁺ T cells recognize peptides which have 8 to 11 amino acids, in most cases presented by major histocompatibility complex (MHC) class I molecules. However, identification of virus-specific CD8⁺ T-cell epitopes remains a complicated and time-consuming process. Various strategies have been developed to define CTL epitopes so far. One of the most common practices to determine immunodominant CTL epitopes on a large scale is based on screening and functional analysis of overlapping 15- to 20-mer peptides covering an entire viral proteome or a given set of immunogenic proteins (19, 23, 32). However, peptides identified through this method are too long to be naturally processed CTL epitopes, and the definition of MHC class I restriction of these peptides still requires further analysis. Rapid and efficient strategies should be developed for the determination of naturally presented CTL epitopes in the context of any given HLA allele of interest. Furthermore, no other HLA alleles except HLA-A2-restricted CTL epitopes have been reported for SARS-CoV-derived proteins (16, 22, 31, 43, 46, 47, 49). This is primarily because of the limitation of the experimental methods for the other HLA alleles. HLA-A24 is one of the most common HLA-A alleles throughout the world, especially in East Asia, where SARS-CoV emerged, second only to HLA-A2 (30). The development of a fast and valid method to screen and identify HLA-A24-restricted epitopes would greatly contribute to the understanding of the specific CTL epitope-stimulated response and widen the application of the epitope-based vaccine among a more universal population (17). A genomewide scanning of HLA binding peptides from SARS-CoV has been performed by Sylvester-Hvid and colleagues, through which dozens of peptides with major HLA supertypes, including HLA-A24 binding capability, have been identified (41).There are strong indications that different peptide ligands, such as peptides with distinct immunodominance, can elicit a diverse specific T-cell repertoire, and even subtle changes in the same peptide can have a profound effect on the response (25, 44). Furthermore, a broader T-cell receptor (TCR) repertoire to a virus-specific peptide-MHC complex can keep the host resistant to the virus and limit the emergence of virus immune-escape mutants (29, 34, 38). Recent studies have demonstrated that the diversity of the selected TCR repertoire (designated as T-cell receptor bias) is clearly influenced by the conformational characteristics of the bound peptide in the MHC groove. Peptides with a flat, featureless surface when presented by MHC generate only limited TCR diversity in a mature repertoire, while featured peptides with exposed residues (without extreme bulges) protruding outside the pMHC landscapes are rather associated with the more diverse T-cell repertoire (15, 28, 39, 44, 45). Therefore, being able to determine the binding features of a peptide to MHC and describe the peptide-MHC topology will help us understand the immunodominance of a given peptide and demonstrate the peptide presentation strategy of the host.Structural proteins of SARS-CoV, such as spike, membrane, and nucleocapsid (N), have been demonstrated as factors of the antigenicity of the virus, as compared with the nonstructural proteins (12, 20). Coronavirus nucleocapsid (N) protein is a highly phosphorylated protein which not only is responsible for construction of the ribonucleoprotein complex by interacting with the viral genome and regulating the synthesis of viral RNA and protein, but also serves as a potent immunogen that induces humoral and cellular immunity (13, 14, 26, 48). The CD8⁺ T-cell epitopes derived from SARS-CoV N protein defined so far mainly cluster in two major immunogenic regions (4, 21, 23, 31, 32, 43). One of them, residues 219 to 235, comprises most of the N protein-derived minimal CTL epitopes identified so far—N220-228, N223-231, N227-235, etc.—all of which are HLA-A*0201 restricted (4, 43). The other region, residues 331 to 365, also includes high-immunogenicity peptides that can induce memory T-lymphocyte responses against SARS-CoV (21, 23, 32). However, until now, no minimal CTL epitope with a given HLA allele restriction has been investigated in this region.Here, based on previously defined immunogenic regions derived from SARS-CoV N protein (21), we identified an HLA-A*2402-restricted epitope, N1 (residues 346 to 354), in the region through a distinct strategy using structural and functional approaches. The binding affinity with HLA-A*2402 molecules and the cellular immunogenicity of the peptide were demonstrated in a series of assays. The X-ray crystal structure of HLA-A*2402 complexed with peptide N1 has shown a novel host strategy to present an immunodominant CTL epitope by intrachain hydrogen bond as a featured epitope.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide

Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2003 as a significant threat to human health, and CoVs still represent a leading source of novel viruses for emergence into the human population. The CoV spike (S) protein mediates both receptor binding (via the S1 domain) and membrane fusion (via the S2 domain) and shows many features of a class I fusion protein, including the presence of distinct heptad repeats within the fusion domain (37). A critical feature of any viral fusion protein is the so-called “fusion peptide,” which is a relatively apolar region of 15 to 25 amino acids that interacts with membranes and drives the fusion reaction (9, 34, 38). Fusion peptides can be classified as N-terminal or internal, depending on their location relative to the cleavage site of the virus fusion protein (23). One key feature of viral fusion peptides is that within a particular virus family, there is high conservation of amino acid residues; however, there is little similarity between fusion peptides of different virus families (26). Despite these differences, some common themes do emerge, including a high level of glycine and/or alanine residues, as well as critical bulky hydrophobic amino acids. In several cases, the fusion peptide is known to contain a central “kink.” In the case of influenza virus hemagglutinin (HA), which is a classic example of an N-terminal fusion peptide, the N- and C-terminal parts of the fusion peptide (which are α-helical) penetrate the outer leaflet of the target membrane, with the kink at the phospholipid surface. The inside of the kink contains hydrophobic amino acids, with charged residues on the outer face (18). Internal fusion peptides (such as Ebola virus [EBOV] GP) often contain a conserved proline near their centers but also require a mixture of hydrophobic and flexible residues similar to N-terminal fusion peptides (9, 11). It is believed that the kinked fusion peptide sits in the outer leaflet of the target membrane and possibly induces positive curvature to drive the fusion reaction (22). It is important to note that, despite the presence of key hydrophobic residues, viral fusion peptides often do not display extensive stretches of hydrophobicity and can contain one or more charged residues (8). Ultimately, fusion peptide identification must rely on an often complex set of criteria, including structures of the fusion protein in different conformations, biophysical measurements of peptide function in model membranes, and biological activity in the context of virus particles.To date, the exact location and sequence of the CoV fusion peptide are not known (4); however, by analogy with other class I viral fusion proteins, it is predicted to be in the S2 domain. Overall, three membranotropic regions in SARS-CoV S2 have been suggested as potential fusion peptides (14, 17). Based on sequence analysis and a hydrophobicity analysis of the S protein using the Wimley-White (WW) interfacial hydrophobic interface scale, initial indications were that the SARS-CoV fusion peptide resided in the N-terminal part of HR1 (heptad repeat 1) (5, 6), which is conserved across the Coronaviridae. Mutagenesis of this predicted fusion peptide inhibited fusion in syncytia assays of S-expressing cells (28). This region of SARS-CoV has also been analyzed by other groups in biochemical assays (16, 17, 29) and defined as the WW II region although Sainz et al. (29) actually identified another, less conserved and less hydrophobic, region (WW I) as being more important for fusion. Peptides corresponding to this region have also been studied in biochemical assays by other groups (13). In addition, a third, aromatic region adjacent to the transmembrane domain (the membrane-proximal domain) has been shown to be important in SARS-CoV fusion (15, 20, 25, 30). This membrane-proximal domain likely acts in concert with a fusion peptide in the S2 ectodomain to mediate final bilayer fusion once conformational changes have exposed the fusion peptide in the ectodomain. To date, there is little or no information on the fusion peptides of CoVs other than SARS-CoV, except for the identification of the N-terminal part of the mouse hepatitis virus (MHV) S HR1 domain as a putative fusion peptide based on sequence analysis (6). In none of these cases (for SARS-CoV or MHV) is the role of these sequences as bone fide fusion peptides established.The majority of class I fusion proteins prime fusion activation by proteolytic processing, with the cleavage event occurring immediately N-terminal to the fusion peptide (21). In the case of SARS-CoV, early reports analyzing heterologously expressed SARS-CoV spike protein indicated that most of the protein was not cleaved (31, 39) but that there was some possibility of limited cleavage at the S1-S2 boundary (39). However, it is generally considered that S1-S2 cleavage is not directly linked to fusion peptide exposure in the case of SARS-CoV or any other CoV (4). Recently, however, it has been shown that SARS-CoV S can be proteolytically cleaved at a downstream position in S2, at residue 797 (2, 36). Here, we investigated whether cleavage at this internal position in S2 might expose a domain with properties of a viral fusion peptide. We carried out a mutagenesis study of SARS-CoV S residues 798 to 815 using cell-cell fusion and pseudovirus assays, as well as lipid mixing and structural studies of an isolated peptide, and we show the importance of this region as a novel fusion peptide for SARS-CoV.     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.