Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

The Lipidomes of Vesicular Stomatitis Virus,Semliki Forest Virus,and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.Enveloped viruses acquire their lipid envelope from the membranes of host cells (43). In this process, the nucleocapsid or the nucleocapsid-matrix complex of the viruses buds out of the cell and becomes enveloped by a segment of the host membrane. This membrane segment is modified during the budding process, such that virally encoded membrane proteins are included in the viral envelope, while most host proteins are excluded. Since viruses usually do not carry lipid-synthesizing enzymes, the lipids in the viral envelope are derived from the host membrane. The lipid compositions of enveloped viruses have been studied for years (2, 15, 17, 18, 23, 25, 34, 36, 38, 40). One question that remains to be answered is whether the lipids are included passively, and thus the lipid composition of the envelope reflects the lipid composition of the host membrane, or whether lipid sorting occurs, leading to selective inclusion of some lipids and exclusion of others. This issue has been complicated by the fact that the lipid bilayer is no longer considered a homogenous liquid but contains fluctuating nanoscale assemblies of sphingolipids, saturated phospholipids, cholesterol (Chol), and proteins, called lipid rafts (13, 44). Lipid rafts can be induced to coalesce—usually by protein-protein interactions—into larger, dynamic platforms that function in signal transduction, intracellular membrane transport, and other membrane functions (45). It was also proposed that viruses make use of these membrane domains during their exit from cells (29, 32).A major complication in comparing viral envelopes with host cell membranes is the difficulty in obtaining host cell membranes of purity similar to that of the easily purified viruses. Many studies are faulted by the impurity of the cell membranes analyzed. Moreover, the early work in this field employed conventional analytical methods (such as thin-layer chromatography) that provide only semiquantitative estimates of the total abundance of the major lipid classes. Most importantly, lipid species diversity could not be analyzed. Recent developments in mass spectrometry (MS) have enabled comprehensive and quantitative analyses of lipidomes at the level of individual molecular species. The lipidomes of human immunodeficiency virus (HIV), murine leukemia virus (6, 7), and several bacteriophages (20, 21) were recently analyzed by these new methods.This paper focuses on two well-characterized enveloped viruses, Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV). SFV is an RNA virus belonging to the Togaviridae family of the Alphaviridae that acquires its envelope by budding from the host cell plasma membrane (PM) (46). Early studies analyzed the lipid composition of the viral envelope and also that of the host cell PM (39, 40). These studies revealed strong similarity between the envelope of SFV and the host PM, but one important discrepancy was the higher Chol-to-phospholipid ratio in the virus.VSV is an RNA virus belonging to the Rhabdoviridae family and also hijacks its envelope from the host cell PM (35), but the lipid specificity of the budding process remains controversial. The most recent studies claim that VSV buds from localized regions that do not reflect the average composition of the PM (23, 36). It has also been claimed that lipid rafts are involved in VSV envelope assembly during budding (37).We used BHK-21 cells as host cells to purify SFV and VSV. The purposes of this study were (i) to establish a robust, comprehensive, and quantitative method to analyze lipidomes, including the full complement of glycerolipid, glycerophospholipid, and sphingolipid species as well as Chol; (ii) to establish a protocol for purification of PM suitable for MS analysis; and (iii) to analyze and compare the lipidomes of SFV, VSV, and the BHK-21 PM.We found that the lipidomes of SFV and VSV are similar in molecular composition and are closely related to that of the BHK-21 PM. The small differences observed could be explained by the high degrees of curvature generated during the viral budding process.     

Genome Comparison of a Nonpathogenic Myxoma Virus Field Strain with Its Ancestor,the Virulent Lausanne Strain

One of the best-studied examples of host-virus coevolution is the release of myxoma virus (MV) for biological control of European rabbits in Australia and Europe. To investigate the genetic basis of MV adaptation to its new host, we sequenced the genome of 6918, an attenuated Spanish field strain, and compared it with that of Lausanne, the strain originally released in Europe in 1952. Although isolated 43 years apart, the genomes were highly conserved (99.95% identical). Only 32 of the 159 MV predicted proteins revealed amino acid changes. Four genes (M009L, M036L, M135R, and M148R) in 6918 were disrupted by frameshift mutations.Myxoma virus (MV), the causative agent of myxomatosis, belongs to the Leporipoxvirus genus of the Poxviridae family (9). Two distinct types of MV have been identified: South American MV, which circulates in Sylvilagus brasiliensis, and Californian MV, which circulates in Sylvilagus bachmani. Each virus is highly adapted to its host, causing a benign cutaneous fibroma at the site of inoculation. Both types of MV infect the European rabbit (Oryctolagus cuniculus), causing myxomatosis. The Californian strain MSW is more virulent for European rabbits than South American strains such as SLS or Lausanne (54). Another leporipoxvirus, Shope fibroma virus (SFV), is found in eastern North America in Sylvilagus floridanus. SFV protects European rabbits against myxomatosis (24), and it is routinely used as a vaccine.One of the best-studied examples of host-virus coevolution is the use of MV for biological control of European rabbits (22, 23, 29). It is particularly unusual because the precise time the virus was released is known, and the original viruses are available for comparison with current strains. MV (the SLS strain) was deliberately released in Australia in 1950 and soon after (1952) in France (the Lausanne strain), whence it rapidly spread across Europe, and it has become endemic since then. For almost 60 years, a complex coevolution of host and virus has occurred, characterized by the emergence of attenuated viral strains and rabbits selected for resistance to MV (11, 12, 30).The MV Lausanne strain and SFV have been completely sequenced (13, 61). MV encodes 171 genes, versus 165 encoded by SFV. The genetic information is highly conserved between the two viruses. Recently, preliminary sequencing of the MSW strain indicated that the major genomic differences with the Lausanne strain localize at the left terminal end of the MSW genome (31). In MSW, the terminal inverted repeats (TIRs) are extended, causing the duplication of five complete open reading frames (ORFs), which are present as a single copy near the right TIR in the Lausanne strain (9). To date, little molecular analysis concerning the adaptation of MV to its new host has been performed. Studies involving Australian field strains found small differences with reference to the SLS and Lausanne strains (49, 50), suggesting that adaptation (and the concomitant attenuation) of MV is not associated with major genetic changes such as large deletions. This finding is in contrast to what has been reported for attenuated poxviruses obtained by extensive cell culture passaging, which usually present substantial genomic deletions or rearrangements (5, 25, 36, 47, 48).Strain 6918 is a naturally attenuated MV isolated in Spain in 1995 (7). It is therefore a descendant of the Lausanne strain recovered after 43 years of continuous evolution in the field. It has been used for the development of a “transmissible vaccine” intended to protect wild-rabbit populations against both myxomatosis and rabbit hemorrhagic disease virus (RHDV) in Spain, where the European rabbit plays a key role in the Mediterranean ecosystems (18). For this purpose, a recombinant virus, 6918VP60-T2, was constructed by inserting the capsid gene of RHDV into the genome of strain 6918 (4, 6, 7, 56, 57). The genomes of 6918 and 6918VP60-T2 have been sequenced. Here we report the results of our comparison of the genomic sequences of Lausanne and 6918. To our knowledge, this is the first comparative genomic analysis involving two poxvirus field strains linked by a clearly recorded lineage, one being fully virulent and the other virtually nonpathogenic. The results provide relevant insights into the mechanisms of MV attenuation that occurred as a consequence of the adaptation of the virus to its new host.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

Poxviruses produce complement regulatory proteins to subvert the host''s immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host''s immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement''s role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE''s regulatory capacity. These results suggest that EMICE''s role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.Poxviruses encode in their large double-stranded DNA genomes many factors that modify the immune system (30, 56). The analysis of these molecules has revealed a delicate balance between viral pathogenesis and the host''s immune response (2, 21, 31, 61). Variola, vaccinia, monkeypox, cowpox, and ectromelia (ECTV) viruses each produce an orthologous complement regulatory protein (poxviral inhibitor of complement enzymes [PICE]) that has structural and functional homology to host proteins (14, 29, 34, 38, 41, 45, 54). The loss of the regulatory protein resulted in smaller local lesions with vaccinia virus lacking the vaccinia virus complement control protein (VCP) (29) and in a greater local inflammatory response in the case of cowpox lacking the inflammation-modulatory protein (IMP; the cowpox virus PICE) (35, 45, 46). Additionally, the complete loss of the monkeypox virus inhibitor of complement enzymes (MOPICE) may account for part of the reduced mortality observed in the West African compared to Congo basin strains of monkeypox virus (12).The complement system consists of proteins on the cell surface and in blood that recognize and destroy invading pathogens and infected host cells (36, 52). Viruses protect themselves from the antiviral effects of complement activation in a variety of ways, including hijacking the host''s complement regulatory proteins or producing their own inhibitors (7, 8, 15, 20, 23). Another effective strategy is to incorporate the host''s complement regulators in the outermost viral membrane, which then protects the virus from complement attack (62). The extracellular enveloped virus (EEV) produced by poxviruses acquires a unique outer membrane derived from the Golgi complex or early endosomes that contain the protective host complement regulators (58, 62). Poxviruses have multiple infectious forms, and the most abundant, intracellular mature virions (IMV), are released when infected cells lyse (58). The IMV lacks the outermost membrane found on EEV and is sensitive to complement-mediated neutralization. The multiple strategies viruses have evolved to evade the complement system underscore its importance to innate and adaptive immunity (15, 36).The most well-characterized PICE is VCP (24-29, 34, 49, 50, 53, 55, 59, 60). Originally described as a secreted complement inhibitor (34), VCP also attaches to the surface of infected cells through an interaction with the viral membrane protein A56 that requires an unpaired N-terminal cysteine (26). This extra cysteine also adds to the potency of the inhibitor by forming function-enhancing dimers (41). VCP and the smallpox virus inhibitor of complement enzymes (SPICE) bind heparin in vitro, and this may facilitate cell surface interactions (24, 38, 50, 59). The coevolution of variola virus with its only natural host, humans, likely explains the enhanced activity against human complement observed with SPICE compared to the other PICEs (54, 64).Our recent work with ECTV, the causative agent of mousepox infection, demonstrated that the classical and alternative pathways of the complement system are required for host survival (48). The mouse-specific pathogen ECTV causes severe disease in most strains and has coevolved with its natural host, analogous to variola virus in humans (9). This close host-virus relationship is particularly important for evaluating the role of the complement system, given the species specificity of many complement proteins, receptors, and regulators (10, 47, 62). Additionally, the availability of complement-deficient mice permits dissection of the complement activation pathways involved. Naïve C57BL/6 mouse serum neutralizes the IMV of ECTV in vitro, predominately through opsonization (48). Maximal neutralization requires natural antibody, classical-pathway activation, and amplification by the alternative pathway. C3 deficiency in the normally resistant C57BL/6 strain results in acute mortality, similar to immunodeficiencies in important elements of the antiviral immune response, including CD8⁺ T cells (19, 32), natural killer cells (18, 51), and gamma interferon (33). During ECTV infection, the complement system acts in the first few hours and days to delay the spread of infection, resulting in lower levels of viremia and viral burden in tissues (48).This study characterized the PICE produced by ECTV, ectromelia virus inhibitor of complement enzymes (EMICE), and assessed its complement regulatory activity. Recombinant EMICE (rEMICE) decreased activation of both human and mouse complement. Murine cells produced EMICE at 4 to 6 h postinfection prior to the release of the majority of the complement-sensitive IMV from infected cells. rEMICE protected ECTV IMV from complement-mediated neutralization. Further, EMICE produced during natural infection inhibited complement deposition on infected cells by the alternative pathway. ECTV likely produces this abundance of EMICE to protect both the IMV and infected cells.     

Andes Virus Disrupts the Endothelial Cell Barrier by Induction of Vascular Endothelial Growth Factor and Downregulation of VE-Cadherin

Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infection. High levels of virus replication occur in microvascular endothelial cells but without a virus-induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus-induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease.Hantaviruses, of the family Bunyaviridae, are rodent-borne RNA viruses. Members of the Hantavirus genus have been identified as etiologic agents of two severe human diseases: hemorrhagic fever with renal syndrome (HFRS), which is caused by the Old World hantaviruses, and hantavirus pulmonary syndrome (HPS), which is caused by the New World hantaviruses (38, 39). Sin Nombre virus (SNV) and Andes virus (ANDV) are the main causes of HPS in the Americas. The major hantavirus target in humans is the microvascular endothelium, and the basis of HPS and HFRS is attributed to microvascular leakage (9, 34, 57). Common clinical features of HPS are interstitial pneumonitis with variable amounts of mononuclear cell infiltration, congestion, and both interstitial and alveolar edema (4, 34, 57). Despite the prominent accumulation of viral antigen in the infected vascular endothelium, no evidence of cellular destruction has been observed (57). Absence of a cytopathic effect has also been reported in in vitro studies of hantavirus infection of human primary endothelial cells (35, 46). In general, it is believed that induction of an uncontrolled immune response to the hantavirus infection, rather than the viral infection per se, is the cause of the microvascular leakage and ultimately HPS and HFRS (3, 48, 57). So far, a limited number of in vitro permeability studies have reported either no significant changes in the vascular permeability upon hantavirus infection or a significant increase only when mediators of increased permeability are exogenously added to the hantavirus-infected cells (12, 22, 46).Endothelial cell permeability is a highly regulated process and is maintained by both tight and adherens junctions (47). The disruption of adherens junctions is sufficient to disturb the endothelium barrier function and cause an increase in permeability and formation of edema (25, 47). Adherens junctions are largely composed of vascular endothelial (VE) cadherin (VE-cadherin), an endothelial cell-specific member of the cadherin family of adhesion protein (51, 52). Adherens junctions and in particular VE-cadherin are targets of the signaling pathway of agents that increase vascular permeability (7, 8, 10). Vascular endothelial growth factor (VEGF), one of the most potent vascular permeability agents, exerts its effects after binding to its homologous membrane tyrosine kinase receptor, VEGF-R2, whose expression is restricted to endothelial cells. It is known that VEGF-R2 interacts with VE-cadherin, and together they maintain the endothelial cell barrier (26). When VEGF is present, it binds to VEGF-R2, and that initiates the internalization and degradation of VE-cadherin and disruption of the adherens junctions (10, 54).In general, increase of vascular permeability is an important component of severe disease progression in hemorrhagic fevers (36). A number of studies have investigated the cause of increased vascular permeability in viral hemorrhagic fevers induced by viruses such as Dengue virus or Ebola virus (41, 42, 50, 53, 56). Studies of vascular permeability during hantavirus infection in vitro have mainly been performed in the presence of various inflammatory agents and growth factors (12, 15, 19, 22, 46). A recent study demonstrated that pathogenic hantaviruses sensitize the endothelium and cause hyperpermeability in response to high levels of exogenously added VEGF (12). We show here that VE-cadherin downregulation can be observed in ANDV-infected cells in the absence of exogenous VEGF. The downregulation of VE-cadherin in the absence of exogenous VEGF led us to the discovery that endothelial cells infected with ANDV induce the production of VEGF at early times postinfection. The early increased secretion of VEGF coincides with the initiation of downregulation of the adherent junction protein VE-cadherin and an increase in permeability of endothelial cells. The involvement of VEGF-R2 in VE-cadherin downregulation was demonstrated by antibody blockage of VEGF-R2 that resulted in significant recovery of VE-cadherin levels. These data indicate that the increased vascular permeability seen in HPS could be a direct result of hantavirus infection of the endothelium and may occur through a pathway involving VEGF-induced downregulation of VE-cadherin at early times postinfection.     

Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34⁺ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE^D) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ^−/− mouse model engrafted with human CD34⁺ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.Dengue is a mosquito-borne flavivirus infection that has emerged as a serious public health problem worldwide. Four serotypes of dengue virus (DEN-1 to DEN-4) are capable of causing human disease varying in severity from acute self-limiting febrile illness to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The plasma leakage, hemorrhagic manifestations, and shock that characterize DHF/DSS are considered to have an immunological basis, as they are more common during secondary infection with a heterologous dengue virus strain (15, 28, 33). However, severe clinical manifestations can also occur during primary dengue infection, pointing to a contributory role of viral virulence factors. The WHO estimates that more than 20,000 people worldwide, mainly children, die each year from serious complications of dengue. No specific antiviral therapies are currently available for treating the infection, and efforts to develop a safe prophylactic vaccine have been hindered by the complex role of the immune system in disease pathogenesis (39, 52, 57). Thus, novel treatment strategies that block viral replication and/or to attenuate the exaggerated cytokine response associated with DHF/DSS complications are urgently needed.Potent and specific gene silencing mediated by RNA interference (RNAi) has generated a great deal of interest in development of RNAi as a therapeutic strategy against viral infections (50, 54). Many studies have demonstrated the effectiveness of the RNAi approach to suppress flavivirus infection, including dengue virus replication in experimental cell lines (3, 23, 26, 42, 60). In addition, the versatility of RNAi could also be exploited to block important host mediators that contribute to dengue pathogenesis. However, the existence of four distinct dengue virus serotypes and the ability of viruses to develop resistance to RNAi by mutating their sequences will have to be taken into account before clinical use can be contemplated. A more serious hurdle for RNAi therapeutics is the specific delivery of small interfering RNA (siRNA) to relevant cell types.Even though dengue virus antigens have been detected in many tissues, including liver, spleen, lymph node, and skin of patients with DHF/DSS, macrophages and dendritic cells (DCs) are considered the predominant infected cell types (9, 36, 59). Following the bite of an infected Aedes mosquito, the initial local viral replication is believed to take place in the skin DCs, including myeloid DCs and Langerhans cells (31, 53, 59). Dengue-infected DCs play a key role in the immunopathogenesis of DHF/DSS, as, along with macrophages, they release proinflammatory cytokines and soluble factors that mediate plasma leakage, thrombocytopenia, and hypovolemic shock associated with severe dengue infection (14, 15, 29, 38). Therefore, development of a method to introduce siRNA into DCs would be an important step toward using RNAi therapeutically to suppress viral replication and/or to attenuate the vigorous host cytokine responses in dengue infection (7, 19).To target DCs, we used a previously characterized 12-amino-acid peptide identified from a phage display peptide library that specifically binds to a ligand expressed on DCs (10). In an earlier study, we demonstrated that fusing nucleic acid-binding nine d-arginine residues to a neuronal cell-targeting peptide enabled siRNA delivery to neuronal cells (27). Here, in a similar approach, we synthesized a chimeric peptide consisting of the DC-targeting peptide fused to nona-D-arginines (9dR) to target siRNA selectively to DCs. We investigated whether the DC3-9dR peptide could deliver siRNA targeting a dengue virus envelope sequence to reduce the viral load in DCs. As tumor necrosis factor alpha (TNF-α) is one of the acute-phase cytokines with a major role in inducing plasma leakage in dengue infection (8, 12, 17, 20), we also explored the possibility of reducing TNF-α expression in DC in vitro and in vivo. Our findings demonstrate the potential of a targeted RNAi-based approach for simultaneously decreasing viral load and reducing aberrant cytokine responses in DCs.     

Characterization of a Putative Ancestor of Coxsackievirus B5

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3D^pol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.     

gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.Through correlative studies with macaques challenged with simian immunodeficiency virus (SIV), the initial targets of infection in nontraumatic vaginal exposure to human immunodeficiency virus type 1 (HIV-1) have been identified as subepithelial T cells and dendritic cells (DCs) (18, 23, 31, 36-38). While human transmission may differ from macaque transmission, the existing models of human transmission remain controversial. For the virus to successfully reach its CD4⁺ targets, HIV must first traverse the columnar mucosal epithelial cell barrier of the endocervix or uterus or the stratified squamous barrier of the vagina or ectocervix, whose normal functions include protection of underlying tissue from pathogens. This portion of the human innate immune defense system represents a significant impediment to transmission. Studies have placed the natural transmission rate of HIV per sexual act between 0.005 and 0.3% (17, 45). Breaks in the epithelial barrier caused by secondary infection with other sexual transmitted diseases or the normal physical trauma often associated with vaginal intercourse represent one potential means for viral exposure to submucosal cells and have been shown to significantly increase transmission (reviewed in reference 11). However, studies of nontraumatic exposure to SIV in macaques demonstrate that these disruptions are not necessary for successful transmission to healthy females. This disparity indicates that multiple mechanisms by which HIV-1 can pass through mucosal epithelium might exist in vivo. Identifying these mechanisms represents an important obstacle to understanding and ultimately preventing HIV transmission.Several host cellular receptors, including DC-specific intercellular adhesion molecule-grabbing integrin, galactosyl ceramide, mannose receptor, langerin, heparan sulfate proteoglycans (HSPGs), and chondroitin sulfate proteoglycans, have been identified that facilitate disease progression through binding of HIV virions without being required for fusion and infection (2, 3, 12, 14, 16, 25, 29, 30, 43, 46, 50). These host accessory proteins act predominately through glycosylation-based interactions between HIV envelope (Env) and the host cellular receptors. These different host accessory factors can lead to increased infectivity in cis and trans or can serve to concentrate and expose virus at sites relevant to furthering its spread within the body. The direct transcytosis of cell-free virus through primary genital epithelial cells and the human endometrial carcinoma cell line HEC1A has been described (7, 9); this is, in part, mediated by HSPGs (7). Within the HSPG family, the syndecans have been previously shown to facilitate trans infection of HIV in vitro through binding of a specific region of Env that is moderately conserved (7, 8). This report also demonstrates that while HSPGs mediate a portion of the viral transcytosis that occurs in these two cell types, a significant portion of the observed transport occurs through an HSPG-independent mechanism. Other host cell factors likely provide alternatives to HSPGs for HIV-1 to use in subverting the mucosal epithelial barrier.gp340 is a member of the scavenger receptor cysteine-rich (SRCR) family of innate immune receptors. Its numerous splice variants can be found as a secreted component of human saliva (34, 41, 42) and as a membrane-associated receptor in a large number of epithelial cell lineages (22, 32, 40). Its normal cellular function includes immune surveillance of bacteria (4-6, 44), interaction with influenza A virus (19, 20, 32, 51) and surfactant proteins in the lung (20, 22, 33), and facilitating epithelial cell regeneration at sites of cellular inflammation and damage (27, 32). The secreted form of gp340, salivary agglutinin (SAG), was identified as a component of saliva that inhibits HIV-1 transmission in the oral pharynx through a specific interaction with the viral envelope protein that serves to agglutinate the virus and target it for degradation (34, 35, 41). Interestingly, SAG was demonstrated to form a direct protein-protein interaction with HIV Env (53, 54). Later, a cell surface-associated variant of SAG called gp340 was characterized as a binding partner for HIV-1 in the female genital tract that could facilitate virus transmission to susceptible targets of infection (47) and as a macrophage-expressed enhancer of infection (10).     

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.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.