Vesicular Stomatitis Virus Envelope Research Articles

Vesicular stomatitis virus (VSV) as a vaccine vector for immunization against viral infections

Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, is a promising candidate for potential use in construction of antiviral vaccines. In the natural environment VSV is a pathogen of wild ungulates and livestock. Some of the features that make VSV an excellent platform for the development of a range of viral therapeutics includes its immunogenicity and ability to grow to high titers in cell lines approved for vaccine use. Infection in humans is rare and usually asymptomatic, with mild flu-like symptoms. Moreover, due to affinity of VSV envelope glycoprotein to the LDL (low-density lipoprotein) receptor, VSV is effective at targeting a variety of tissues in vivo. A series of research results confirm the possibility of developing VSV-based vaccines against human papilloma viruses (HPV), human immunodeficiency virus (HIV), hepatitis B virus (HBV) and filoviruses (MARV, ZEBOV and SEBOV), as well as the potential use of a successfully developed vaccine against hepatitis C virus (HCV). VSV is neurotropic and infection can cause a viral encephalitis in experimental animals. Therefore, intensive studies are being undertaken to achieve satisfactory expression of the viral antigens while maintaining the safety of the constructed vectors.

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

BackgroundThis study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.MethodologyThree series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.Principal FindingsMMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.Conclusions/SignificanceThese results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.

Efficient transduction of healthy and malignant plasma cells by lentiviral vectors pseudotyped with measles virus glycoproteins

A lot of genes deregulated in malignant plasma cells (PCs) involved in multiple myeloma have been reported these last years. The expression of some of these genes is associated with poor survival. A critical step is to elucidate the biological mechanisms triggered by these gene products. Such studies are hampered by the difficulty to obtain malignant PCs and to genetically modify them. Usual lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein poorly transduced healthy and malignant PCs. Here, we report that LVs pseudotyped with the hemagglutinin and fusion glycoproteins from the measles Edmonston strain (H/F-LVs) can efficiently and stably transduce healthy and primary malignant PCs, without modifying their main phenotypic characteristics. Both LV pseudotypes efficiently transduced human myeloma cell lines. Importantly, both healthy and malignant PCs expressed CD46 and SLAMF1/CD150 membrane proteins, which are critical receptors for binding and productive genetic modification by H/F-LVs. The ability to efficiently introduce and express a given gene into PCs opens the possibility to study in detail PC biology.

Baculovirus GP64-mediated entry into mammalian cells.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability

The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146–150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein. Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral DNA, although less efficiently than WT. Collectively, these findings demonstrate that the linker region is essential for proper assembly and stability of cores and efficient replication.

A lentiviral vector pseudotype suitable for vaccine development

Lentiviral vectors (LV) are promising vaccines because they transduce dendritic cells (DC) in vivo. To translate LV vaccines into clinical trials, bulk production will be necessary. The present study aimed to find a suitable envelope for LV vaccine production from stable packaging cells because the commonly used vesicular stomatitis virus envelope (VSV-G) is cytotoxic. The envelope from Ross river virus (RRV) was selected. It can infect mouse and human cells, allowing testing in animals before clinical translation. We used VSV-G for comparison. Vectors produced with each envelope were titred on human 293T cells and mouse 3T3 cells. RRV-pseudotyped LV (RRV-LV) infected mouse myeloid DC in culture and immunized mice. An approximately 50-fold higher dose of RRV-LV than VSV-G-LV was required to generate a similar T cell response. The RRV-LV could also be used to infect human mDC and to prime a human T cell immune response. RRV envelope is a suitable candidate to be used for the construction of an LV producer cell line. LV vaccines with RRV envelope can be tested in mice and in human immune cell cultures. The higher dose of RRV-LV required for vaccine efficacy compared to VSV-G-LV will partly be offset by ease of production.

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

Attenuation of lymphocytic choriomeningitis virus relaxes interferon-dependence of adaptive immune responses and of virus control (44.12)

Abstract Interferons (IFNs) are essential for CD8+ T cell responses against lymphocytic choriomeningitis virus (LCMV) and for humoral responses against vesicular stomatitis virus (VSV), and they have a profound impact on control of these infections. Recently, we have developed LCM viruses carrying the VSV envelope gene (rLCMV/VSVG) as live-attenuated arenavirus vaccines. Here, we assessed how innate and adaptive immunity contribute to control of rLCMV/VSVG. Even in mice lacking IFN type I and II receptors, rLCMV/VSVG was efficiently controlled and induced neutralizing antibodies at titers similar or even higher than in wildtype mice. Similarly, significantly higher frequencies of specific CD8+ T cells were detected which remained protective for more than 400 days. Mice lacking both IFN type I receptor and RAG1 (AR) failed to control the virus and became viremic while RAG1 single-deficient mice exhibited circulating viral RNA but not overt viremia. Type II IFN was found to have an effect on rLCMV/VSVG control only on an AR background, while NK cells did not seem to contribute. Thus, viral attenuation for vaccine use was found to considerably relax the IFN-dependence of virus control and of antiviral adaptive immune responses. This provides a better understanding of the attenuated behavior of these vaccine candidates and highlights the varying impact of IFN in LCMV infection.

A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts.

BackgroundCytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry.ResultsThe method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies.ConclusionGiven the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines

Large-scale production of gene therapeutics comprising equine infectious anaemia virus (EIAV) -based lentiviral vectors (LVs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome expression constructs). As the vesicular stomatitis virus (VSV-G) envelope protein is cytotoxic, its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet repressor (TetR) system was selected to regulate expression of VSV-G and Gag/Pol, necessitating the introduction of a fourth construct, encoding TetR, into the cell line. We have generated an inducible packaging cell line that shows tight control of the packaging components, and high-titre vector production on transient transfection of the EIAV genome. The cell line is stable for at least 7 weeks in the absence of selective pressure. To verify that this packaging cell line can support the generation of producer cell lines it was transfected stably with an EIAV genome cassette encoding ProSavin; a gene therapeutic for Parkinson's disease. Producer cell lines were generated, which on induction, yielded ProSavin with titres comparable to the transient system.

Lentiviral Transfection of Ependymal Primary Cultures Facilitates the Characterisation of Kinocilia-specific Promoters

Ependymal primary cultures (EPCs) are an established model for studying ependymal cell biochemistry and the biology of kinocilia-bearing cells. However, the difficulty in causing them to express transgenes at high efficiency has been an important drawback of the system. Indeed plasmid-based transfection attempts remain at an efficiency below 1% and fail to elicit reporter gene expression, namely green fluorescent protein (GFP) synthesis, in any of the kinocilia-bearing cells of the cultures. Human immunodeficiency virus pseudotyped with the vesicular stomatitis virus envelope glycoprotein (HIV/VSV-G) and encoding GFP under the control of the ubiquitously recognised promoter of elongation factor 1 alpha (EF1alpha) also does not cause transgene expression in the kinocilia-bearing cells of an EPC when applied at multiplicities of infection (MOIs) of up to 40 and destroys the culture when the MOI is increased further. In contrast, HIV/VSV-G encoding GFP under the control of a promoter specifically active in kinocilia-bearing cells leads to transgene expression in up to 79% of the kinociliated cells of an EPC when applied at an MOI of 20. This has permitted the initial characterisation of the promoter for the gene specifically transcribed in kinocilia-bearing cells, wdr16. The results have identified two regions of 100 nucleotides length each, which are critical for promoter activity and contain putative binding sites for the transcription factors Foxd1, Sox17 and Spz1. It appears that wdr16 is controlled by a bidirectional promoter also responsible for regulating the syntaxin 8 gene.

The Chemokine Receptor CXCR4 and the Metalloproteinase MT1-MMP Are Mutually Required during Melanoma Metastasis to Lungs

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.

Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

BackgroundGene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon.MethodsWe analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters.ResultsWe found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1α) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV.ConclusionTaken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Transduction Efficiency of Pantropic Retroviral Vectors Is Controlled by the Envelope Plasmid to Vector Plasmid Ratio

Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.

VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability

BackgroundThe machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined.ResultsHere, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity.ConclusionDefects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.

Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.