Nonpermissive Cells Research Articles

Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor

HIV-1 virus infectivity factor (Vif) is one of the four accessory proteins that are characteristic of primate lentiviruses and critically required for the infection of host cells. Vif plays a key role in replication and transmission of the virus in non-permissive cells, such as primary T cells and macrophages. Using co-precipitation and co-fractionation techniques, evidence has been provided that Vif interacts with a variety of host proteins, such as the cytidine deaminases APOBEC3G and 3F, the Cullin5/EloBC ubiquitin-ligase complex, Fyn and Hck tyrosine kinases, as well as with viral components, such as the immature Gag precursor and viral RNA. We report on the expression, purification and molecular characterization of a folded recombinant subtype C Vif. Vif was expressed in E. coli with a C-terminal hexahistidine tag and purified by nickel affinity chromatography. We obtained approximately 5mg protein per liter of bacterial culture, with a purity >95%. The expected molecular mass of 23.7kDa was confirmed by mass spectrometry. Although dynamic light scattering and small angle X-ray scattering measurements revealed the presence of high molecular weight aggregates in the protein preparation, circular dichroism analysis showed that the protein contains mainly folded β-sheet elements and exhibits remarkable thermal stability (T (m)>95°C). Recombinant Vif may be used as a tool to study its biological functions and tertiary structure, as well as for the development of diagnostic, therapeutic and preventive strategies for HIV-1 infections.

Functional analysis of Spodoptera litura nucleopolyhedrovirus p49 gene during Autographa californica nucleopolyhedrovirus infection of SpLi-221 cells

The Spodoptera litura nucleopolyhedrovirus (SpltNPV) antiapoptotic gene Splt-p49 is able to rescue replication of a p35-null Autographa californica nucleopolyhedrovirus (AcMNPV) in AcMNPV-permissive Sf9 cells. In this study, an AcMNPV p35 knockout bacmid was generated through ET homologous recombination in Escherichia coli and designated as vAc(P35-KO). The Splt-p49 gene was transposed into the polyhedrin locus of vAc(P35-KO) to investigate if Splt-p49 has any antiapoptotic activity in the context of AcMNPV infection of AcMNPV-nonpermissive SpLi-221 cells. Our results demonstrated that Splt-p49 could not inhibit the apoptosis induced by AcMNPV infection of SpLi-221 cells when it was under the control of its native promoter. Western blot analysis showed that Splt-P49 was poorly expressed. When the expression of Splt-P49 was under the control of Drosophila hsp70 promoter, the expression of Splt-P49 was advanced, and a higher level of Splt-P49 was detected. As a result, the apoptosis of SpLi-221 cells was inhibited; however, budded-virus production did not improve in comparison with that in AcMNPV-infected SpLi-221 cells. These data indicated that there are other barrier(s) to AcMNPV replication in the nonpermissive SpLi-221 cells besides apoptosis.

The infection and viral DNA replication ofBocavirusminute virus of canine in permissive cells and non-permissive cells

The infection and viral DNA replication of<i>Bocavirus</i>minute virus of canine in permissive cells and non-permissive cells

Gene Expression and Transcription Factor Profiling Reveal Inhibition of Transcription Factor cAMP-response Element-binding Protein by γ-Herpesvirus Replication and Transcription Activator

Herpesvirus replication involves the expression of over 80 viral genes in a well ordered sequence, leading to the production of new virions. Viral genes expressed during the earliest phases of replication often regulate both viral and cellular genes. Therefore, they have the potential to bring about dramatic functional changes within the cell. Replication and transcription activator (RTA) is a potent immediate early transcription activator of the gamma-herpesvirus family. This family includes Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, human pathogens associated with malignancy. Here we combine gene array technology with transcription factor profiling to identify the earliest DNA promoter and cellular transcription factor targets of RTA in the cellular genome. We find that expression of RTA leads to both activation and inhibition of distinct groups of cellular genes. The identity of the target genes suggests that RTA rapidly changes the cellular environment to counteract cell death pathways, support growth factor signaling, and also promote immune evasion of the infected cell. Transcription factor profiling of the target gene promoters highlighted distinct pathways involved in gene activation at specific time points. Most notable throughout was the high level of cAMP-response element-binding protein (CREB)-response elements in RTA target genes. We find that RTA can function as either an activator or an inhibitor of CREB-response genes, depending on the promoter context. The association with CREB also highlights a novel connection and coordination between viral and cellular "immediate early" responses.

Hepatitis C Virus Infection of Neuroepithelioma Cell Lines

Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication. We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism. Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas nonpermissive neural cell lines lacked CLDN1 and, in some cases, SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1, and HCV E2. Furthermore, anti-CD81, interferon, and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells. Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at levels comparable to those of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.

A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

Branched oligomerization of cell-permeable peptides markedly enhances the transduction efficiency of adenovirus into mesenchymal stem cells

Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only approximately 60% of MSCs were maximally transduced at 500 muM of monomeric CPPs, >95% of MSCs were transduced with 0.1 muM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.

Role of the C-terminal domain of the HIV-1 glycoprotein in cell-to-cell viral transmission between T lymphocytes

BackgroundMutant HIV (HIV-Env-Tr712) lacking the cytoplasmic tail of the viral glycoprotein (Env-CT) exhibits a cell-type specific replication phenotype such that replicative spread occurs in some T-cell lines (referred to as permissive cells) but fails to do so in most T-cell lines or in PBMCs (referred to as non-permissive cells). We aim to gain insight on the underlying requirement for the Env-CT for viral spread in non-permissive cells.ResultsWe established that in comparison to HIV-Wt, both cell-free and cell-to-cell transmission of mutant HIV-Env-Tr712 from non-permissive cells were severely impaired under naturally low infection conditions. This requirement for Env-CT could be largely overcome by using saturating amounts of virus for infection. We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype. Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells.ConclusionsDuring natural infection conditions, the HIV-Env-CT is critically required for viral transmission in cultures of non-permissive cells by both cell-free and cell-to-cell routes and is instrumental for Gag accumulation to the VS. The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

Vif of Feline Immunodeficiency Virus from Domestic Cats Protects against APOBEC3 Restriction Factors from Many Felids

To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

Abstract 2592: Host dependency factors for binding and internalization of oncolytic Seneca Valley Virus

Abstract Background Seneca Valley Virus (SVV-001), an experimental oncolytic picornavirus, is the prototype member of the genus Senecavirus of the family Picornaviridae. SVV-001 is remarkable for its exquisite tumor selectivity for human cancers with neuroendocrine features including small cell lung carcinoma (SCLC) and numerous pediatric cancers while being non-cytotoxic in normal human cells in vitro and in vivo. The mechanism of tropism and neuroendocrine tumor specificity of SVV-001 has not been elucidated previously. Identification of host factors involved in infection would have immediate benefit for ongoing clinical trials by providing potential biomarker candidates to predict efficacy. Here we present data implicating α4 integrin as a key component of SVV-001 infection and demonstrating that the virus is dependent on caveloae for internalization and trafficking. Results Microarray analysis comparing permissive with non-permissive cell lines and membrane sub-proteome analysis of permissive H446 SCLC cells predict α4 integrin is a potential receptor candidate for SVV-001. To test SVV-001 dependency on α4 integrin, stable shRNA knockdown cell lines were generated from the permissive parental SCLC cell line H446. Viral infection was studied by single virus particle tracking, GFP reporter virus infection, and FACS analysis. α4 integrin knockdown cells showed reduced viral internalization and intracellular motility and were resistant to infection. Heterologous expression of human α4 integrin in chinese hamster ovary (CHO) cells significantly increased binding of fluorescently-labeled virus to the cell surface, but did not result in productive infection. Tracking of fluorescently-labeled SVV-001 in permissive cells expressing either clathrin-GFP, caveolin-1-GFP, or flotillin-1-GFP revealed a striking and long-lasting colocalization of internalized viral particles with caveolin-rich vesicles. Dependency on caveolin-1 for internalization was supported by results from pharmacological inhibition experiments. Pre-treatment of cells with nystatin, a cholesterol sequestration drug, resulted in a reduction in virus-induced cytopathic effect when compared to untreated cells. Stable shRNA caveolin-1 knockdown cells showed significantly reduced internalization, reduced infection by recombinant GFP reporter virus and significant impairment in SVV-001 genomic RNA release. Conclusions Defining the mechanism of tropism for SVV-001 is a key factor in successful clinical application. α4 integrin and caveolin-1 represent the first host cell factors identified in the mechanism of tropism of SVV-001 leading to a basic understanding of how the virus binds and enters permissive cells. These findings may have direct relevance for current clinical trials of SVV-001 in adult and pediatric settings and for development of second-generation derivatives of this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2592.

Canarypox and fowlpox viruses as recombinant vaccine vectors: an ultrastructural comparative analysis

Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.

Interaction between single wall carbon nanotubes and a human enteric virus

Activated single wall carbon nanotubes have been used for biomedical purposes as carriers for drugs, peptides, proteins and nucleic acids. A large volume of data speaks to their suitability to act as a carrier. The ability of two differently activated SWNTs (with carboxyl groups or with carboxyl groups and polyethylenimine (PEI)) to form a complex with the hepatitis A virus was evaluated. Both types of activations permitted the formation of a virus–SWNT complex. However, their patterns were different. The carboxyl-activated nanotubes had a somewhat low adsorptive capacity that was related inversely to the concentrations of the SWNTs and viruses. Statistical analysis, using the χ 2-test, showed no significant differences between the SWNT–PEI ratios of 1:2.5, 1:1 and 1:0.5. The addiction of PEI improved the adsorption, probably because of the electropositive charge of the molecule. Adsorption was optimal between 100 μg and 10 ng with a SWNTs–PEI weight ratio of 1:0.2 up to an inoculum of 10 5 genome equivalents of hepatitis A virus. Reducing or increasing this weight ratio reduced the adsorptive capacity of the PEI, and this adsorption activity was time and contact-dependent. Thus, SWNTs coated with PEI are able to complex with viruses, and they might be used in the future to transfect non-permissive cell lines.

Characterization of transgene expression in adenoviral vector-based HIV-1 vaccine candidates

Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies. More recently, the capability of inducing potent cell-mediated and humoral immunity has made these vectors equally attractive candidates for prophylactic or therapeutic vaccine applications. Merck and Co., Inc., developed HIV-1 vaccine candidates based on adenovirus serotype 5 (Ad5) vectors in which the E1 gene, a critical component for adenovirus replication, was replaced by the cytomegalovirus immediate/early promoter, followed by mutated versions of the HIV-1 gag, pol or nef genes (constructs referred to as MRKAd5gag, MRKAd5pol and MRKAd5nef, respectively). Vaccine performance was evaluated in vitro in a novel assay that measures the level of transgene expression in non-permissive A549 cells. Various combinations of vectors were studied. The results indicate that the vaccine induces a dose-dependent expression of the HIV-1 transgenes in vitro. Furthermore, the gag, pol, and nef transgenes are expressed differentially in A549 cells in an MOI-dependent and formulation-dependent manner, yielding an unexpected enhancement of protein expression in trivalent vs. monovalent formulations. Our data suggest that the presence of additional virus in multivalent formulations increases individual transgene expression in A549 cells, even when the amount of DNA encoding the gene of interest remains constant. This enhancement appears to be controlled at the transcriptional level and related to both the total amount of virus and the combination of transgenes present in the formulation.

Point mutations in BHV-1 Us3 gene abolish its ability to induce cytoskeletal changes in various cell types

The Us3 gene is conserved among alphaherpesviruses and codes for a protein kinase, a multifunctional protein involved in many phases of virus infection, like nuclear egress, modulation of apoptosis and modification of the cellular cytoskeleton. Bovine herpesvirus (BHV-1), a member of the Alphaherpesvirinae, contains an open reading frame homologous to Us3 of other herpesviruses, which has been identified as a serine/threonine kinase (Takashima, Y., Tamura, H., Xuan, X., Otsuka, H., 1999. Identification of the Us3 gene product of BHV-1 as a protein kinase and characterization of BHV-1 mutants of the Us3 gene. Virus Res. 59, 23–34). To study the activity of BHV-1 Us3, we have cloned its sequence under control of the human cytomegalovirus (HCMV) promoter/enhancer and introduced it into a recombinant baculovirus (Bac Us3). Confocal microscopy analysis showed profound cytoskeletal modifications in various BHV-1-permissive and non-permissive cells transduced with BacUs3. We observed that Us3 expression changed cellular shape and induced formation of long microtubule-containing cell projections, a phenomenon which had also been observed in cells expressing pseudorabies virus Us3. The intracellular localization of Us3 was mostly nuclear but when the protein accumulated it could be detected in the cytoplasm, cell membranes and projections. Mutated forms of BHV-1 Us3 with point mutations near or within the kinase catalytic domain did not affect cell morphology indicating that kinase activity of BHV-1 Us3 is required for its cytoskeleton remodelling function.

Coupling of receptor interference and a host-dependent post-binding entry deficiency in a gammaretroviral envelope protein

BackgroundSL3-2 is a unique polytropic murine gammaretroviral isolate that is only able to infect murine cells. We have previously shown that two mutations R212G and T213I located on the surface of the receptor binding domain in a region designated the VR3 loop can alter the species tropism of this envelope protein. This location suggests that the VR3 loop composition has an influence on receptor interaction and thereby affects binding as well as superinfection resistance. In order to investigate this further, we have studied the binding and interference patterns of the SL3-2 envelope and its mutants.ResultsWe find unexpectedly that wild type SL3-2 envelope binds equally well to both permissive and non-permissive cells, indicating a post binding defect when interacting with the human Xpr1. Using replication competent viruses containing envelopes from SL3-2 or its mutants we find that the same amino acid mutations can dramatically alter the interference profile of this polytropic ENV, suggesting that the same amino acid changes that cause the post binding defect also influence interaction with the receptor.ConclusionsThe envelope protein of SL3-2 MLV shows an entry defect on non-murine cells. This is coupled to a dramatically reduced ability to interfere with entry of other polytropic viruses. Two point mutations in the VR3 loop of the receptor binding domain of this envelope result both in a much increased interference ability and in removing the post-binding defect on non-murine cells, suggesting that both of these phenotypes are a consequence of insufficient interaction between the envelope and the receptor

CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway

As we have recently found a novel oncogene, the cancer-upregulated gene 2 (CUG2), which was elevated in a variety of tumor tissues such as the ovary, liver, lung and pancreas, we examined whether reovirus could efficiently induce cytolysis in cancer cells expressing CUG2 and thus be used as a potential cancer therapeutic agent. In this study, we describe experiments in which we use reovirus to treat NIH3T3 cells stably expressing either CUG2 (NIH-CUG2) or vector only (NIH-Vec). NIH-CUG2 cells readily support reoviral proliferation and undergo apoptosis, whereas NIH-Vec cells are highly resistant to reoviral infection and virus-induced apoptosis. This notable result may be explained by the observation that CUG2 expression inhibits PKR activation, leading to reoviral proliferation in nonpermissive NIH3T3 cells. Furthermore, reovirus infection results in almost complete regression of tumorgenic NIH-CUG2 cells in transplanted nude mice. As we found that CUG2 enhances activation of MAPK (ERK, JNK and p38), Src kinase and Ras, we examined whether CUG2 confers reoviral replication independent of the Ras or p38 MAPK signaling pathway. From these experiments we found that either inhibition of p38 MAPK or Ras blocks reoviral proliferation even in the presence of CUG2 but inhibition of ERK, JNK and Src kinase does not, indicating that activation of p38 MAPK and Ras has critical roles in reoviral replication in CUG2-expressing tumor cells. Accordingly, we propose that reovirus can be useful in the treatment of transformed cells expressing CUG2, which is commonly detected in various tumor tissues.

Contribution of the porcine aminopeptidase N (CD13) receptor density to porcine epidemic diarrhea virus infection

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), which belong to group 1 coronaviruses, are important viral pathogens in pigs causing lethal diarrhea. As with the other members in the group 1, theses viruses are also known to use the host aminopeptidase N (APN) as the major receptor for cell entry. Remarkably, it was found that they utilize distinct cultured cell lines for in vitro virus propagation, since PEDV could not be replicated in swine testis (ST) cells expressing native porcine APN (pAPN), which are highly susceptible to TGEV. To explain the mechanism causing this discrimination, we postulated that there may be a correlation between the pAPN expression level and PEDV infection. As a first step toward understanding the role of cellular receptor density in PEDV replication, therefore, sub-lines of ST cells stably overexpressing recombinant pAPN were generated. We initially confirmed that the control ST cells do express relatively low levels of endogenous pAPN. In contrast, in the engineered stable cell lines, a high level of recombinant pAPN expression was demonstrated. The introduction of a pAPN gene into nonpermissive ST cells was further found to be fully sufficient to support productive infection, revealing that constitutive overexpression of pAPN can directly rescue PEDV multiplication. We further assessed whether the pAPN enzymatic function is relevant to PEDV infection. The enzymatic active motif-null mutant pAPN still retained the ability to exert its receptor activity and consequently, to cause infectious virus production. Moreover, the only APN inhibitor blocking the protease activity site had no obvious negative effect on viral infection, indicating that the enzymatic role of APN is dispensable for the process of virus replication. Taken together, our results suggest that pAPN receptor density appears to be an important factor in contributing to efficient PEDV infection.

Porcine reproductive and respiratory syndrome virus attachment is mediated by the N-terminal domain of the sialoadhesin receptor

Sialoadhesin (Sn) is an important receptor for viral attachment and internalization of porcine reproductive and respiratory syndrome virus (PRRSV) to porcine alveolar macrophages (PAM). To investigate whether the N-terminal domain of Sn is sufficient and/or necessary for PRRSV attachment, we constructed a series of truncated fragments of porcine Sn and expressed these in the non-permissive PK15 cell line. The first 150 amino acids comprising the entire first domain of the Sn N-terminal region was necessary for PRRSV binding to cells, and the N-terminal domain alone was sufficient for virus attachment. The attachment of PRRSV to PAM cells was inhibited by polyclonal anti-serum against the N-terminal region of porcine Sn in a dose-dependent manner. The present study demonstrates that the first domain at the N-terminus of Sn mediates PRRSV attachment to PAM cells and contributes to better understanding the interaction between PRRSV and its host cells.

Identification of the Cellular Prohibitin 1/Prohibitin 2 Heterodimer as an Interaction Partner of the C-Terminal Cytoplasmic Domain of the HIV-1 Glycoprotein

Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3′UTR than for the 5′UTR, even though this region contained at least one high affinity Vif binding site (apparent Kd = 27 ± 6 nM). Several Vif binding sites were identified in 5′ and 3′UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5′UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.