Plasma Membrane Of Infected Cells Research Articles

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy. While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.

Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.

The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40's roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD-NTD to form a dimer, dimer-to-dimer NTD-NTD oligomerization to form a hexamer, and end-to-end hexamer CTD-CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD-CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD-NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD-CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.

Membrane Binding of HIV-1 Accessory Protein Nef on Sparsely-Tethered Bilayer Lipid Membranes: An Spr Study

Human Immunodeficiency Virus-1 Nef is an accessory protein essential for the progression of HIV-1 infection by aiding viral production by down-regulating defense mechanisms of the infected cell to help evade immune response. Nef has been shown to interact with Src- and Tec-family kinases at the plasma membrane of infected cells, leading to constitutive kinase activation. For example, blocking Interleukin-2-inducable T-cell Kinase (Itk) activity with pharmacological inhibitors has been shown to result in decreased viral spread. Evidence also shows that Nef dimerizes on the membrane as it recruits kinases. However, the exact mechanism of this interaction between Nef and the kinases, or indeed between those two classes of proteins and the membrane, is not much understood. We used Surface Plasmon Resonance to study the binding of Nef on sparsely-tethered bilayer lipid membranes (stBLMs) in preparation for future studies in complex with kinases. Nef exhibits two distinct binding modes, one fast and one slow, which we attribute to two different membrane binding sites on Nef. The fast binding mode is likely that of the expected binding mechanism for Nef; insertion into the membrane of a myristoyl tail, whereby the core region of the protein is distant from the membrane surface. The second binding mode has Nef in much closer proximity to the membrane, with a much higher surface coverage. This in turn blocks the fast binding mode in subsequent concentration additions. For a given concentration, the binding rate is far higher when that concentration is added directly to a neat bilayer, rather than reached through a series of concentration additions. Previous Neutron Reflectometry (NR) characterization of membrane-bound Nef showed a concentration-dependent conformation change, consistent with this interpretation.

Evidence for an Interaction between Influenza Hemagglutinin and PIP2

The influenza viral protein hemagglutinin (HA) forms dense nanoscale clusters on the plasma membrane of infected cells. HA clusters mediate fusion, which is crucial to infectivity. Previous studies have shown a connection between influenza infection and phosphoinositide-related cellular processes; however, no interaction between HA and the major plasma membrane phosphoinositide PIP2 (phosphatidylinositol-(4,5)-biphosphate) has yet been shown. Super-resolution imaging in fixed cells reveals that HA and PIP2 clusters are colocalized with similar radial distribution functions. Furthermore, single particle tracking experiments in living cells indicate that PIP2 molecules have reduced mobility and increased confinement in the presence of HA clusters. This evidence suggests an underlying interaction between HA and PIP2 and merits further investigation. Understanding cellular interactions with influenza viral proteins may lead to new anti-viral therapies that are strain independent.

Molecular Mechanism of Externalization of Phosphatidylserine on the Surface of Ebola Virus Particles.

Ebola virus (EBOV) is an enveloped filamentous virus that causes severe hemorrhagic fever in humans and nonhuman primates with up to 90% fatality. Accumulating evidence indicates that various viruses, including EBOV, exploit the host apoptotic clearance machinery to enhance their entry into host cells by externalizing phosphatidylserine (PS) in the viral envelope. PS is typically distributed in the inner layer of the plasma membrane (PM) in normal cells. Progeny EBOV virions bud from the PM of infected cells, suggesting that PS is likely flipped to the outer leaflet of the envelope of Ebola virions. Currently, the intracellular dynamics of PS during EBOV infection are poorly understood. This review summarizes recent progress in determining the molecular mechanism of externalization of PS in the envelope of EBOV particles. We also discuss future directions and how viral apoptotic mimicry could be targeted for therapeutics.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

Cooperation of the Ebola Virus Proteins VP40 and GP1,2 with BST2 To Activate NF-κB Independently of Virus-Like Particle Trapping.

BST2 is a host protein with dual functions in response to viral infections: it traps newly assembled enveloped virions at the plasma membrane in infected cells, and it induces NF-κB activity, especially in the context of retroviral assembly. In this study, we examined whether Ebola virus proteins affect BST2-mediated induction of NF-κB. We found that the Ebola virus matrix protein, VP40, and envelope glycoprotein, GP, each cooperate with BST2 to induce NF-κB activity, with maximal activity when all three proteins are expressed. Unlike human immunodeficiency virus type 1 Vpu protein, which antagonizes both virion entrapment and the activation of NF-κB by BST2, Ebola virus GP does not inhibit NF-κB signaling even while it antagonizes the entrapment of virus-like particles. GP from Reston ebolavirus, a nonpathogenic species in humans, showed a phenotype similar to that of GP from Zaire ebolavirus, a highly pathogenic species, in terms of both the activation of NF-κB and the antagonism of virion entrapment. Although Ebola virus VP40 and GP both activate NF-κB independently of BST2, VP40 is the more potent activator. Activation of NF-κB by the Ebola virus proteins either alone or together with BST2 requires the canonical NF-κB signaling pathway. Mechanistically, the maximal NF-κB activation by GP, VP40, and BST2 together requires the ectodomain cysteines needed for BST2 dimerization, the putative BST2 tetramerization residue L70, and Y6 of a potential hemi-ITAM motif in BST2's cytoplasmic domain. BST2 with a glycosylphosphatidylinositol (GPI) anchor signal deletion, which is not expressed at the plasma membrane and is unable to entrap virions, activated NF-κB in concert with the Ebola virus proteins at least as effectively as wild-type BST2. Signaling by the GPI anchor mutant also depended on Y6 of BST2. Overall, our data show that activation of NF-κB by BST2 is independent of virion entrapment in the case of Ebola virus. Nonetheless, BST2 may induce or amplify proinflammatory signaling during Ebola virus infection, potentially contributing to the dysregulated cytokine response that is a hallmark of Ebola virus disease.IMPORTANCE Understanding how the host responds to viral infections informs the development of therapeutics and vaccines. We asked how proinflammatory signaling by the host protein BST2/tetherin, which is mediated by the transcription factor NF-κB, responds to Ebola virus proteins. Although the Ebola virus envelope glycoprotein (GP1,2) antagonizes the trapping of newly formed virions at the plasma membrane by BST2, we found that it does not inhibit BST2's ability to induce NF-κB activity. This distinguishes GP1,2 from the HIV-1 protein Vpu, the prototype BST2 antagonist, which inhibits both virion entrapment and the induction of NF-κB activity. Ebola virus GP1,2, the Ebola virus matrix protein VP40, and BST2 are at least additive with respect to the induction of NF-κB activity. The effects of these proteins converge on an intracellular signaling pathway that depends on a protein modification termed neddylation. Better mechanistic understanding of these phenomena could provide targets for therapies that modulate the inflammatory response during Ebola virus disease.

Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes.

The mumps virus (MuV) small hydrophobic protein (SH) is a type I membrane protein expressed in infected cells. SH has been reported to interfere with innate immunity by inhibiting tumor necrosis factor alpha (TNF-α)-mediated apoptosis and NF-κB activation. To elucidate the underlying mechanism, we generated recombinant MuVs (rMuVs) expressing the SH protein with an N-terminal FLAG epitope or lacking SH expression due to the insertion of three stop codons into the SH gene. Using these viruses, we were able to show that SH reduces the phosphorylation of IKKβ, IκBα, and p65 as well as the translocation of p65 into the nucleus of infected A549 cells. Reporter gene assays revealed that SH interferes not only with TNF-α-mediated NF-κB activation but also with IL-1β- and poly(I·C)-mediated NF-κB activation, and that this inhibition occurs upstream of the NF-κB pathway components TRAF2, TRAF6, and TAK1. Since SH coimmunoprecipitated with tumor necrosis factor receptor 1 (TNFR1), RIP1, and IRAK1, we hypothesize that SH exerts its inhibitory function by interacting with TNFR1, interleukin-1 receptor type 1 (IL-1R1), and TLR3 complexes in the plasma membrane of infected cells.IMPORTANCE The MuV SH has been shown to impede TNF-α-mediated NF-κB activation and is therefore thought to contribute to viral immune evasion. However, the mechanisms by which SH mediates NF-κB inhibition remained largely unknown. In this study, we show that SH interacts with TNFR1, IL-1R1, and TLR3 complexes in infected cells. We thereby not only shed light on the mechanisms of SH-mediated NF-κB inhibition but also reveal that SH interferes with NF-κB activation induced by interleukin-1β (IL-1β) and double-stranded RNA.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.

Exercising Restraint

Ten years ago, while in Paul Bieniasz's group, I determined that an interferon-induced antiviral activity inhibited the release of enveloped viruses by tethering them to the plasma membrane of infected cells. This commentary examines the lead up to this study and how our observations led to the identification of tetherin.

Membrane Interactions of the Mason-Pfizer Monkey Virus Matrix Protein and Its Budding Deficient Mutants

Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction.The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the 1H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.

Asymmetric structure of the influenza A virus and novel function of the matrix protein M1.

Influenza virus is an enveloped virus. It comprises two major modules: external lipoprotein envelope and internal ribonucleoprotein (RNP) containing the genomic negative-strand RNA. Lipoprotein envelope contains four vital proteins: hemagglutinin (HA), neuraminidase (NA), transmembrane ionic channel M2, and minor amounts of nuclear export protein NEP. RNP contains RNA and four polypeptides: major nucleocapsid protein NP and three polymerase subunits PB1, PB2, PA. Both modules are linked with each other by matrix M1 maintaining the virus integrity. According to the structural function, NP and M1 are predominant in virus particle in the amounts of 1000 and 3000 molecules, respectively. In addition to the structural function, M1 plays a role in regulation of intracellular and nuclear migration of viral RNP and virus assembly, referred as budding process, at the plasma membrane in infected cells. The bipolar structure of the influenza virus characterized by asymmetric location of RNP and nonregular distribution of M1 and M2 inside the virion is reviewed. The role of M1 in maintaining the asymmetric structure of the virus particle and regulation of RNP transport inside virus particle is considered. First experimental data confirming (i) intravirion RNP transport and its outside exit directed by the M1 and (ii) the importance of this process in virus uncoating and initiation of infection in target cell are discussed. A novel class of antiviral agents activating ATP-ase of the early endosome compartment in the target cell is discussed.

Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities.

The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.

Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells.

Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions.

ESCRTs are everywhere.

The ESCRT proteins are an ancient system that buds membranes and severs membrane necks from their inner face. Three "classical" functions of the ESCRTs have dominated research into these proteins since their discovery in 2001: the biogenesis of multivesicular bodies in endolysosomal sorting; the budding of HIV-1 and other viruses from the plasma membrane of infected cells; and the membrane abscission step in cytokinesis. The past few years have seen an explosion of novel functions: the biogenesis of microvesicles and exosomes; plasma membrane wound repair; neuron pruning; extraction of defective nuclear pore complexes; nuclear envelope reformation; plus-stranded RNA virus replication compartment formation; and micro- and macroautophagy. Most, and perhaps all, of the functions involve the conserved membrane-neck-directed activities of the ESCRTs, revealing a remarkably widespread role for this machinery through a broad swath of cell biology.

Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry.

The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Intercellular transmission of HTLV-1: not all mechanisms have been revealed

HTLV-1 is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL) and of HTLV-1-Associated Myelopathy/tropical spastic paraparesis (HAM/TSP). It is mainly detected in CD4+ lymphocytes in vivo, but proviral genomes have also been detected although less frequently, in CD8+ T lymphocytes, B lymphocytes, monocytes, macrophages, dendritic cells and other non-lymphoid cells. Virus spread is highly dependent on cell-cell contact. This mode of transmission is correlated with an increased ability of infected cells to migrate, a property linked to cytoskeleton reorganization induced by the viral Tax protein. Cell-to-cell transmission relies on at least three non-exclusive molecular pathways. First, a specialized area, the "virological synapse'' (VS) promotes direct transmission of budding HTLV-1 particles into a synaptic cleft formed between infected and uninfected cells. Second, HTLV-1 particles accumulate at the plasma membrane of infected cells in a biofilm-like extracellular viral assembly that resembles a bacterial biofilm. Viral biofilm is rapidly transmitted to uninfected cells when infected cells contact target cells. Finally, membrane extensions called inter-cellular conduits facilitate HTLV-1 proteins transfer from infected to uninfected target cells, and may stabilize cell-cell contacts. The aim of this review is to summarize the molecular mechanisms of these HTLV-1 transmission pathways.

Paradigm Shift in the Mechanism of HIV-1 Core Biogenesis

The proteolytic cleavage of the poly-protein HIV-1 Gag, which is assembled on the surface of plasma membranes of infected cells, drives the conversion of the virus from the initial immature, non-infectious form to the functionally distinct mature, infectious form. Gag cleavage results in a series of structural changes, ultimately leading to the formation of a mature core. Current models for assembly of the mature core suggest that the cleaved HIV capsid protein (CA) nucleates in a concentration-dependent manner, and polymerizes forming the conical core in a diffusion-controlled process. These models also postulate that the core begins to grow at its narrow end, and stops growing once it reaches the membrane at the opposite end. Thus, the size of the virus itself is expected to be the primary factor that determines core size. Our findings challenge this view.Cryo-electron microscopic analyses show that along with infectious viruses, viral isolates also comprise large membranous structures that contain multiple, freely-floating mature cores. Numerous instances of membrane-attached assembly intermediates with partially formed “core-rolls” that are at different stages of conversion from a planar Gag lattice to the mature core are also observed. These results indicate that the mechanism of core formation involves a non-diffusional, cooperative transition triggered by cleavage of the immature Gag lattice, resulting in its rolling away from the plasma membrane to form sheets that wrap around the viral RNA. Unlike the present models, our mechanism predicts that the generation of infectious HIV-1 will be severely affected by incomplete cleavage CA from the Gag matrix (MA), and that even a small percentage of uncleaved MA-CA would prevent the core from rolling away. This prediction explains previous experimental results; only 4% of cleavage-resistant MA-CA is sufficient to cause a 50% reduction of infectivity.