The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ～3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.     

Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes

Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.     

Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta-D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca](i), which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.     

Role of the transmembrane domain of marburg virus surface protein GP in assembly of the viral envelope

The major protein constituents of the filoviral envelope are the matrix protein VP40 and the surface transmembrane protein GP. While VP40 is recruited to the sites of budding via the late retrograde endosomal transport route, GP is suggested to be transported via the classical secretory pathway involving the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network until it reaches the plasma membrane where most filoviral budding takes place. Since both transport routes target the plasma membrane, it was thought that GP and VP40 join there to form the viral envelope. However, it was recently shown that, upon coexpression of both proteins, GP is partially recruited into peripheral VP40-enriched multivesicular bodies, which contained markers of the late endosome. Accumulation of GP and VP40 in this compartment was presumed to play an important role in the formation of the filoviral envelope. Using a domain-swapping approach, we were able to show that the transmembrane domain of GP was essential and sufficient for (i) partial recruitment of chimeric glycoproteins into VP40-enriched multivesicular bodies and (ii) incorporation into virus-like particles (VLPs) that were released upon expression of VP40. Only those chimeric glycoproteins which were targeted to VP40-enriched endosomal multivesicular bodies were subsequently recruited into VLPs. These data show that the transmembrane domain of GP is critical for the mixing of VP40 and GP in multivesicular bodies and incorporation of GP into the viral envelope. Results further suggest that trapping of GP in the VP40-enriched late endosomal compartment is important for the formation of the viral envelope.     

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.     

Inhibition of human immunodeficiency virus envelope glycoprotein- mediated single cell lysis by low-molecular-weight antagonists of viral entry

The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.     

Efficient HIV-1 replication can occur in the absence of the viral matrix protein.

Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells.     

Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability

Cleavage of the rotavirus spike protein, VP4, is required for rotavirus-induced membrane permeability and viral entry into cells. The VP5* cleavage product selectively permeabilizes membranes and liposomes and contains an internal hydrophobic domain that is required for membrane permeability. Here we investigate VP5* domains (residues 248 to 474) that direct membrane binding. We determined that expressed VP5 fragments containing residues 248 to 474 or 265 to 474, including the internal hydrophobic domain, bind to cellular membranes but are not present in Triton X-100-resistant membrane rafts. Expressed VP5 partitions into aqueous but not detergent phases of Triton X-114, suggesting that VP5 is not integrally inserted into membranes. Since high-salt or alkaline conditions eluted VP5 from membranes, our findings demonstrate that VP5 is peripherally associated with membranes. Interestingly, mutagenesis of residue 394 (W-->R) within the VP5 hydrophobic domain, which abolishes VP5-directed permeability, had no effect on VP5's peripheral membrane association. In contrast, deletion of N-terminal VP5 residues (residues 265 to 279) abolished VP5 binding to membranes. Alanine mutagenesis of two positively charged residues within this domain (residues 274R and 276K) dramatically reduced (>95%) binding of VP5 to membranes and suggested their potential interaction with polar head groups of the lipid bilayer. Mutations in either the VP5 hydrophobic or basic domain blocked VP5-directed permeability of cells. These findings indicate that there are at least two discrete domains within VP5* required for pore formation: an N-terminal basic domain that permits VP5* to peripherally associate with membranes and an internal hydrophobic domain that is essential for altering membrane permeability. These results provide a fundamental understanding of interactions between VP5* and the membrane, which are required for rotavirus entry.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

Ebola virus VP40 late domains are not essential for viral replication in cell culture

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.     

Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

Multivesicular bodies as a platform for formation of the Marburg virus envelope

The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.     

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.     

Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4

The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.     

Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40(mut) ). VP40(mut) was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40(mut) was specifically impaired in its ability to recruit nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of nucleocapsids to the viral envelope.     

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.     

Baculovirus VP80 protein and the F-actin cytoskeleton interact and connect the viral replication factory with the nuclear periphery

Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.