Mutations in Both the Surface and Transmembrane Envelope Glycoproteins of the RAV-2 Subgroup B Avian Sarcoma and Leukosis Virus Are Required to Escape the Antiviral Effect of a Secreted Form of the TvbS3 Receptor †.

The subgroup A through E avian sarcoma and leukosis viruses ASLV(A) through ASLV(E) are a group of highly related alpharetroviruses that have evolved to use very different host protein families as receptors. We have exploited genetic selection strategies to force the replication-competent ASLVs to naturally evolve and acquire mutations to escape the pressure on virus entry and yield a functional replicating virus. In this study, evolutionary pressure was exerted on ASLV(B) virus entry and replication using a secreted for of its Tvb receptor. As expected, mutations in the ASLV(B) surface glycoprotein hypervariable regions were selected that knocked out the ability for the mutant glycoprotein to bind the sTvbS3-IgG inhibitor. However, the subgroup B Rous associated virus 2 (RAV-2) also required additional mutations in the C-terminal end of the SU glycoprotein and multiple regions of TM highlighting the importance of the entire viral envelope glycoprotein trimer structure to mediate the entry process efficiently. These mutations altered the normal two-step ASLV membrane fusion process to enable infection.

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

Model of the TVA receptor determinants required for efficient infection by subgroup A avian sarcoma and leukosis viruses.

UNLABELLED: The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed. IMPORTANCE: A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.

The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus.

BACKGROUND: The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/ß) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV.

Cellular entry of retroviruses.

The retrovirus family contains several important human and animal pathogens, including the human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). Studies with retroviruses were instrumental to our present understanding of the cellular entry of enveloped viruses in general. For instance, studies with alpharetroviruses defined receptor engagement, as opposed to low pH, as a trigger for the envelope protein-driven membrane fusion. The insights into the retroviral entry process allowed the generation of a new class of antivirals, entry inhibitors, and these therapeutics are at present used for treatment of HIV/AIDS. In this chapter, we will summarize key concepts established for entry of avian sarcoma and leukosis virus (ASLV), a widely used model system for retroviral entry. We will then review how foamy virus and HIV, primate- and human retroviruses, enter target cells, and how the interaction of the viral and cellular factors involved in the cellular entry of these viruses impacts viral tropism, pathogenesis and approaches to therapy and vaccine development.

Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx.

Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA.

Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

Proviruses selected for high and stable expression of transduced genes accumulate in broadly transcribed genome areas.

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

The interferon-induced gene ISG15 blocks retrovirus release from cells late in the budding process.

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.

Receptor binding and low pH coactivate oncogenic retrovirus envelope-mediated fusion.

Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarely requires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusion mediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yet receptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRV receptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and that this effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. In contrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as well as unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Env fusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently, sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, but substantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating that sHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptor priming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely to be different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understanding of virus fusion and cell entry.

A charged second-site mutation in the fusion peptide rescues replication of a mutant avian sarcoma and leukosis virus lacking critical cysteine residues flanking the internal fusion domain.

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

TOJ3, a v-jun target with intrinsic oncogenic potential, is directly regulated by Jun via a novel AP-1 binding motif.

The TOJ3 gene was originally identified on the basis of its specific activation in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17). Overexpression of TOJ3 induces cellular transformation of embryonic avian fibroblasts, revealing an intrinsic oncogenic potential. Transforming activity has also been demonstrated for MSP58, the human homolog of TOJ3, and oncogenic cell transformation by MSP58 is specifically inhibited by the tumor suppressor PTEN. To investigate the mechanism of aberrant TOJ3 gene activation in jun-transformed fibroblasts, the entire quail TOJ3 gene including 13 exons and the 5' regulatory region was isolated. Functional analyses of the promoter by transcriptional transactivation assays revealed that the specific induction of TOJ3 is mediated by a cluster of three noncanonical AP-1 binding motifs (5'-CAGCTCA-3' or 5'-CACCTCA-3') which share the 3' half-site with the consensus motif (5'-TGA(C)/(G)TCA-3'). Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses showed that Jun binds to these motifs with an affinity similar to that observed for binding to an AP-1 consensus site. Noncanonical binding sites are also present in the chicken and human TOJ3/MSP58 promoter regions. These results confirm and extend the previous observation that TOJ3 represents an immediate effector gene of Jun and may point to an essential role of TOJ3/MSP58 in carcinogenesis involving aberrant AP-1 expression.

Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting.

The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Deltap2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Deltap2b or a chimeric Deltap2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Deltap2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.

Anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) in Rous sarcoma virus-induced tumours in chicken.

The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.

Alternate polypurine tracts affect rous sarcoma virus integration in vivo.

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.

De novo biosynthesis of dihydrosphingosine-1-phosphate by sphingosine kinase 1 in mammalian cells.

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid.

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects.

The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similar to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both DeltaCH1 and DeltaCH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.

Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus.

Retrovirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency virus type 1 (HIV-1) Gag CA (capsid) domain in the immature virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroviruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in immature retrovirus particles.