Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

Foot-and-Mouth Disease Virus Replicates Only Transiently in Well-Differentiated Porcine Nasal Epithelial Cells

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin αvβ6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-β) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-β mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-α mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.Foot-and-mouth disease is an economically important disease caused by foot-and-mouth disease virus (FMDV), a picornavirus, belonging to the genus Aphthovirus and family Picornaviridae. Field strains of FMDV infect cells by attaching to integrin receptors through a highly conserved Arg-Gly-Asp (RGD) tripeptide motif which is located on a surface-exposed loop of VP1 (29). A number of different species of RGD-binding integrin (αvβ1, αvβ3, αvβ6, and αvβ8) have been reported to serve as receptors for infection (8, 22-24). However, αvβ6 is believed to function as the principal receptor for infection in the host (33). FMDV is highly infectious and can become established in susceptible animals by inhalation of relatively small levels of airborne virus (predominantly cattle) or ingestion of contaminated material (primarily pigs) (5). Apart from direct infection through injured skin or abrasion, the epithelial cells of the oral and upper respiratory tract mucosal surfaces are likely to be the first point of contact with the virus. In animals, FMDV undergoes early and rapid replication at the predilection sites in the pharynx and tonsil (5). This is followed by a viremic phase lasting 4 or 5 days, during which the virus spreads to other epithelial tissues and establishes itself at other secondary sites of replication (5). At these secondary sites, vesicles normally form, and the vesicles rupture to produce classical ragged-edge erosions. Although nasal mucosa has been suggested as a primary site of FMDV replication (26), there has been no evidence to support this claim (4). FMDV RNA can be detected in the nasal mucosa and trachea of pigs up to 4 days after infection; however, pathological signs of infection are not observed at these sites (3; P. Dash, unpublished observation).Pigs are known to be more resistant to aerosol infection than cattle, though they excrete larger amounts of virus once infection is established (1, 2, 17, 38). The reason for the apparent reduced susceptibility to aerosol infection of pigs is unclear. Here we have investigated the interaction of FMDV with pig nasal and tracheal epithelial cells using polarized, differentiated three-dimensional (3D) cultures. The results provide insight into the possible reasons why pigs are relatively resistant to FMDV by aerosol challenge.     

Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.     

A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.The compact genomes of viruses relative to those of other infectious agents restrict their ability to encode all proteins required to complete their replication cycles. To circumvent this limitation, viruses often utilize cellular factors or processes to complete essential steps in replication. One group of cellular proteins frequently targeted by viruses are cellular chaperones, which include a diverse set of heat shock proteins (hsps) that normally facilitate cellular protein translation, folding, trafficking, and degradation (18, 64). The connection between viruses and cellular chaperones was originally identified in bacteria, where the Escherichia coli hsp40 and hsp70 homologues, encoded by dnaJ and dnaK, respectively, were identified as bacterial genes essential for bacteriophage λ DNA replication (62). Research over the past 30 years has further revealed the importance of cellular chaperones in viral replication, such that the list of virus-hsp connections is now quite extensive and includes viruses from numerous families with diverse genome structures (4, 6, 7, 16, 19, 20, 23, 25, 40, 41, 44, 51, 54, 60). These studies have demonstrated the importance of cellular chaperones in multiple steps of the viral life cycle, including entry, viral protein translation, genome replication, encapsidation, and virion release. However, the list of virus-hsp connections is likely incomplete. Further studies to explore this particular host-pathogen interaction will shed light on virus replication mechanisms and pathogenesis, and potentially highlight targets for novel antiviral agents.To study the role of cellular chaperones in the genome replication of positive-sense RNA viruses, we use flock house virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae family. The FHV life cycle shares many common features with other positive-sense RNA viruses, including the membrane-specific targeting and assembly of functional RNA replication complexes (37, 38), the exploitation of various cellular processes and host factors for viral replication (5, 23, 60), and the induction of large-scale membrane rearrangements (24, 28, 38, 39). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the structural capsid protein precursor, respectively (1). During active genome replication, FHV produces a subgenomic RNA3 (0.4 kb), which encodes the RNA interference inhibitor protein B2 (12, 29, 32). These viral characteristics make FHV an excellent model system to study many aspects of positive-sense RNA virus biology.In addition to the benefits of a simple genome, FHV is able to establish robust RNA replication in a wide variety of genetically tractable eukaryotic hosts, including Drosophila melanogaster (38), Caenorhabditis elegans (32), and Saccharomyces cerevisiae (46). The budding yeast S. cerevisiae has been an exceptionally useful model host to study the mechanisms of viral RNA replication complex assembly and function with FHV (31, 37, 39, 45, 53, 55, 56, 60) as well as other positive-sense RNA viruses (11). The facile genetics of S. cerevisiae, along with the vast array of well-defined cellular and molecular tools and techniques, make it an ideal eukaryotic host for the identification of cellular factors required for positive-sense RNA virus replication. Furthermore, readily available yeast libraries with deletions and regulated expression of individual proteins have led to the completion of several high-throughput screens to provide a global survey of host factors that impact virus replication (26, 42, 52). An alternative approach with these yeast libraries that reduces the inherently high false-negative rates associated with high-throughput screens is to focus on a select set of host genes associated with a particular cellular pathway, process, or location previously implicated in virus replication.We have utilized such a targeted approach and focused on examining the impact of cytosolic chaperones on FHV RNA replication. Previously, we have shown that the cellular chaperone hsp90 facilitates protein A synthesis in Drosophila cells (5, 23), and the hsp40 encoded by the yeast YDJ1 gene facilitates FHV RNA replication in yeast, in part through effects on both protein A accumulation and function (60). In this report, we further extend these observations by examining FHV RNA accumulation in a panel of yeast strains with deletions of known or hypothesized cytosolic chaperones. We demonstrate that cytosolic chaperones can have either suppressive or enhancing effects on FHV RNA accumulation. In particular, related hsp70 members encoded by the SSA and SSB yeast chaperone families have marked and dramatically divergent effects on both genomic and subgenomic RNA accumulation and viral polymerase synthesis. These results highlight the complexities of the host-pathogen interactions that influence positive-sense RNA virus replication and identify the hsp70 family of cytosolic chaperones as key regulators of FHV replication.     

Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism

Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.     

A Lymph Node-Derived Cytopathic Simian Immunodeficiency Virus Mne Variant Replicates in Nonstimulated Peripheral Blood Mononuclear Cells

Lymph nodes (LNs) are sites of active human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication and disease at both early and late stages of infection. Consequently, variant viruses that replicate efficiently and subsequently cause immune dysfunction may be harbored in this tissue. To determine whether LN-associated SIVs have an increased capacity to replicate and induce cytopathology, a molecular clone of SIV was isolated directly from DNA extracted from unpassaged LN tissue of a pig-tailed macaque (Macaca nemestrina) infected with SIVMne. The animal had declining CD4⁺ T-lymphocyte counts at the time of the LN biopsy. In human CD4⁺ T-cell lines, the LN-derived virus, SIVMne027, replicated with relatively slow kinetics and was minimally cytopathic and non-syncytium inducing compared to other SIVMne clones. However, in phytohemagglutinin-stimulated pig-tailed macaque peripheral blood mononuclear cells (PBMCs), SIVMne027 replicated efficiently and was highly cytopathic for the CD4⁺ T-cell population. Interestingly, unlike other SIVMne clones, SIVMne027 also replicated to a high level in nonstimulated macaque PBMCs. High-level replication depended on the presence of both the T-cell and monocyte/macrophage populations and could be enhanced by interleukin-2 (IL-2). Finally, the primary determinant governing the ability of SIVMne027 to replicate in nonstimulated and IL-2-stimulated PBMCs mapped to gag-pol-vif. Together, these data demonstrate that LNs may harbor non-syncytium-inducing, cytopathic viruses that replicate efficiently and are highly responsive to the effects of cytokines such as IL-2.     

Low Endocytic pH and Capsid Protein Autocleavage Are Critical Components of Flock House Virus Cell Entry

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic γ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the γ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of γ peptides from the virus particle, and (iii) the exposed/released γ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17, 19, 45), RNA replication (2, 3, 37), and specific packaging of the genome (33, 44, 53, 54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29, 57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31, 48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.During cell entry enveloped and nonenveloped viral capsid proteins undergo structural rearrangements that enable the virus to breach the membrane bilayer, ultimately releasing the viral genome or nucleocapsid into the cytoplasm. These entry-related conformational changes have been well characterized for enveloped viruses, which use membrane fusion to cross membrane bilayers (reviewed in reference 59). However, the mechanisms nonenveloped viruses employ to breach cellular membranes are poorly defined. Recently, significant parallels in the mechanisms of cell entry have emerged for a diverse group of nonenveloped viruses. Specifically, programmed capsid disassembly and release of small membrane-interacting peptides appear to be a common theme (reviewed in references 4 and 50).The site of membrane penetration depends upon the route of virus entry into the cell. Viruses can enter host cells via several distinct pathways, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, lipid raft-mediated endocytosis, and macropinocytosis (reviewed in reference 40). The two primary routes of virus entry are clathrin-mediated endocytosis, where viruses encounter an acidic environment, and caveolae-mediated endocytosis, which is pH neutral. Many nonenveloped viruses, including adenovirus (24, 52), parvovirus (6), and reovirus (34, 49), require acidic pH during entry. However, numerous nonenveloped viruses have acid-independent entry mechanisms, including rotavirus (28), polyomavirus (43), simian virus 40 (41, 51), and several members of the picornavirus family (7, 14, 32, 42).Upon reaching the appropriate site of membrane penetration, nonenveloped virus capsid proteins are triggered by cellular factors, such as receptor binding and/or exposure to low pH within endosomes, to undergo conformational changes necessary for membrane interactions. These tightly regulated structural rearrangements may include capsid disassembly, exposure of hydrophobic regions, and/or release of membrane-lytic factors. For example, low pH within endosomes triggers adenovirus capsid disassembly, leading to the release of the membrane lytic protein VI (24, 60). In contrast, poliovirus is activated for membrane penetration by a pH-independent mechanism. Receptor binding triggers the poliovirus capsid to undergo a conformational change, resulting in the exposure of the N terminus of VP1 and the release of VP4 (18, 23), both of which facilitate membrane interactions (20). Notably, even though some viruses, such as reovirus, enter cells via an acidic endocytic pathway, membrane penetration is not acid activated (16), indicating that exposure to low pH and membrane penetration are not always mutual events.The overall simplicity of the FHV capsid, composed of a single gene product, along with the wealth of available high-resolution structural information (reviewed in reference 45) make FHV an ideal candidate for understanding nonenveloped virus entry and infection. FHV, a member of the family Nodaviridae, is a nonenveloped insect virus with a bipartite RNA genome surrounded by an icosahedral protein capsid. The quasi-equivalent T=3 virion (∼300-Å diameter) is initially composed of 180 copies of a single coat precursor protein α (44 kDa). Following capsid assembly the α protein undergoes autocatalytic cleavage to generate two particle-associated cleavage products, a large N-terminal fragment, β (39 kDa), and a small C-terminal fragment, γ (4.4 kDa) (22), creating the infectious virion (46). Mutant FHV particles that do not undergo autocatalytic cleavage, and therefore cannot release the γ peptide, are not infectious (46). It has been hypothesized that these particles are noninfectious because they cannot mediate membrane penetration, but this has never been shown directly.The FHV X-ray structure revealed that the γ peptides were located inside the capsid shell with residues 364 to 385 forming amphipathic helices (19). Subsequent studies showed that the FHV capsid is dynamic, with γ transiently exposed to the exterior of the capsid (11). These findings led to a structure-based model of FHV membrane disruption in which the dynamic γ peptides are reversibly exposed to the surface of the capsid (11), “sampling” the environment until they encounter the appropriate cellular trigger. The virus is then activated to undergo an irreversible conformational change in which the γ helical bundles located at each fivefold axis are externalized and released from the virus particle (17, 19). Upon release, the γ pentameric helical bundles are predicted to insert into and create a local disruption of the membrane bilayer to allow the RNA to enter the cytoplasm (10).While biochemical and structural studies have provided the foundation for a model of FHV cell entry, more rigorous in vivo and in vitro studies are necessary to confirm the ideas put forth in this model. Here, we clarify the route of FHV entry and characterize the tightly regulated events required for FHV membrane penetration. We demonstrate for the first time that low endocytic pH is required for FHV infection, that acidic pH promotes FHV membrane penetration, and that particles exposed to low pH exhibit increased hydrophobicity. In addition, we provide evidence that mutant, cleavage-defective particles are blocked specifically at the membrane penetration step during cell entry. Taken together, these findings offer an experimentally supported model of FHV entry into host cells. In addition, these results add to the accumulating evidence that nonenveloped viruses employ common mechanisms to traverse cellular membranes.     

Virus Specific RNA in Cells transformed by RNA Tumour Viruses

Virus specific RNA comprises 5% of the nuclear RNA and 0.5–1.0% of the cytoplasmic RNA of cells transformed by murine sarcoma viruses. Even cryptically transformed cells which possess no detectable virus or viral proteins synthesize detectable amounts of viral RNA.     

Variation in RNA Virus Mutation Rates across Host Cells

It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10⁻⁶ to 10⁻⁴ substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10⁻⁵ s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

细胞水平狂犬病病毒感染的RNA干扰

以质粒pSilencer2.1-U6 Hygro为基础,设计并构建了针对狂犬病病毒(RV)糖蛋白和核蛋白mRNA的共9个siRNA表达载体,转染BHK-21细胞系后,在潮霉素-B的筛选压力下,获得9个稳定转录相关siRNA的BHK-21细胞株。1000TCID50的RV分别感染24孔板内的上述9株细胞,48h后以直接免疫荧光法检测各株细胞上RV的增殖,结果显示,在经不同siRNA表达载体转染的BHK-21细胞中,RV增殖水平有不同程度下降,RV增殖水平最低者为对照细胞的1%,即RNA干扰效应最高可阻断99%RV的感染。针对其中阻断水平超过90%的靶序列G69和N19,人工合成其双链siRNA,瞬时转染BHK-21细胞后,仍可达到80%以上的感染阻断率。本试验为有效阻断RV早期感染提供了新选择,为通过RNAi研究RV的基因组功能提供了新的依据。     

Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid ( N -acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.     

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.