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The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4 + T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.

Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.

Hepatitis C virus (HCV) is one of the leading causes of chronic liver inflammatory disease (hepatitis), which often leads to more severe diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. Liver fibrosis, in particular, is a major pathogenic consequence of HCV infection, and transforming growth factor β1 (TGF-β1) plays a key role in its pathogenesis. Several HCV proteins have been suggested to either augment or suppress the expression of TGF-β1 by HCV-infected cells. Here, we report that TGF-β1 levels are elevated in HCV-infected hepatocytes cultured in vitro and in liver tissue of HCV patients. Notably, the level of TGF-β1 in media from in vitro -cultured HCV-infected hepatocytes was high enough to activate primary hepatic stellate cells isolated from rats. This indicates that TGF-β1 secreted by HCV-infected hepatocytes is likely to play a key role in the liver fibrosis observed in HCV patients. Moreover, we showed that HCV E2 protein triggers the production of TGF-β1 by HCV-infected cells through overproduction of glucose-regulated protein 94 (GRP94). IMPORTANCE Hepatic fibrosis is a critical step in liver cirrhosis caused by hepatitis C virus infection. It is already known that immune cells, including Kupffer cells, mediate liver fibrosis. Recently, several papers have suggested that HCV-infected hepatocytes also significantly produce TGF-β1. Here, we provide the first examination of TGF-β1 levels in the hepatocytes of HCV patients. Using an HCV culture system, we showed that HCV infection increases TGF-β1 production in hepatocytes. Furthermore, we confirmed that the amount of TGF-β1 secreted by HCV-infected hepatocytes was sufficient to activate primary hepatic stellate cells. To understand the molecular basis of TGF-β1 production in HCV-infected hepatocytes, we used HCV replicons and various stable cell lines. Finally, we elucidated that HCV E2 triggered TGF-β1 secretion via GRP94 mediated NF-κB activation. This study contributes to the understanding of liver fibrosis by HCV and suggests a new potential target (GRP94) for blocking liver cirrhosis in HCV patients.

Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.

In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38 hi , CD10 − , CD19 + , CD20 lo , surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF3 1-179 , ORF3 1-152 , and ORF3 1-107 ) no longer produced the 35K form. An N-terminal truncation mutant (ORF3 51-212 ) and a site-directed mutant (ORF3 T201V ) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3 FL ), ORF3 1-179 , ORF3 1-152 , and ORF3 51-212 interacted with the ORF2 protein, while an ORF3 1-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

The strains of Theiler’s murine encephalomyelitis virus, a picornavirus, are divided into two groups according to their neurovirulence after intracerebral inoculation. The highly virulent GDVII strain causes an acute, fatal encephalomyelitis, whereas the DA strain causes a mild encephalomyelitis followed by a chronic inflammatory demyelinating disease associated with viral persistence. Studies with recombinant viruses showed that the capsid plays the major role in determining these phenotypes. However, the molecular basis for the effect of the capsid on neurovirulence is still unknown. In this paper, we describe a large difference in the patterns of infection of primary neuron cultures by the GDVII and DA strains. Close to 90% of the neurons were infected 12 h after inoculation with the GDVII strain, and the cytopathic effect was complete 24 h postinoculation. In contrast, with the DA strain, viral antigens were not detected in neurons until 24 h postinoculation. Infected neurons accounted for only 2% of the total number of neurons, even 6 days after inoculation. No cytopathic effect was visible, and the cultures could be kept for the same length of time as the noninfected controls. Because the neurovirulence of the GDVII strain has been mapped to the capsid, we examined the role of the capsid in this difference of phenotype. We showed, using recombinant viruses, that the capsid was indeed responsible for the pattern of infection observed in vitro, most likely through its role in viral entry. Thus, the levels of neurovirulence of the GDVII and DA strains correlate with their abilities to infect cultured neurons, and this ability is controlled by the capsid.

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.

Aberrant activation of the B-cell compartment and hypergammaglobulinemia were among the first recognized characteristics of HIV-1-infected patients in the early 1980s. It has been demonstrated previously that HIV-1 particles acquire the costimulatory molecule CD40L when budding from activated CD4 + T cells. In this paper, we confirmed first that CD40L-bearing virions are detected in the plasma from untreated HIV-1-infected individuals. To define the biological functions of virus-associated CD40L and fully characterize its influence on the activation state of B cells, we conducted a large-scale gene expression analysis using microarray technology on B cells isolated from human tonsillar tissue. Comparative analyses of gene expression profiles revealed that CD40L-bearing virions induce a highly similar response to the one observed in samples treated with a CD40 agonist, indicating that virions bearing CD40L can efficiently activate B cells. Among modulated genes, many cytokines/chemokines (CCL17, CCL22), surface molecules (CD23, CD80, ICAM-1), members of the TNF superfamily (FAS, A20, TNIP1, CD40, lymphotoxin alpha, lymphotoxin beta), transcription factors and associated proteins (NFKB1, NFKBIA, NFKBIE), second messengers involved in CD40 signaling (TRAF1, TRAF3, MAP2K1, phosphatidylinositol 3-kinase), and the activation-induced cytidine deaminase (AID) were identified. Moreover, we show that soluble factors induced upon the exposure of B cells to CD40L-bearing virions can exert chemoattractant properties toward CD4 + T cells. We thus propose that a positive feedback loop involving CD40L-bearing HIV-1 particles issued from CD4 + T cells productively infected with HIV-1 play a role in the virus-induced dysfunction of humoral immunity by chronically activating B cells through sustained CD40 signaling.

A structural glycopeptide, gp41, derived from the occluded virus of the baculovirus Autographa californica nuclear polyhedrosis virus was characterized. The peptide specifically bound wheat germ agglutinin but was not recognized by a panel of seven other lectins. Reactivity with wheat germ agglutinin was eliminated by treatment of gp41 with beta-N-acetylglucosaminidase, indicating that N-acetylglucosamine (GlcNAc) was present as terminal residues. gp41 was efficiently galactosylated by galactosyltransferase only in the presence of Nonidet P-40, suggesting that GlcNAc residues are not exposed on the surface of the virion. Metabolic labelling of gp41 with [3H]GlcNAc occurred in the presence of tunicamycin. The carbohydrate was released by alkaline borohydride treatment and comigrated with N-acetylglucosaminitol in descending paper chromatography. The data indicate that gp41 contains single residues of GlcNAc O glycosidically linked to the polypeptide chain. Evidence suggesting that gp41 is located in the region between the envelope membrane and the capsid (defined here as the tegument) of the occluded virus is also presented.

Infection of the mouse with Theiler's virus is one of the best animal models for the study of multiple sclerosis, a chronic demyelinating disease of the human central nervous system. The identification of the virus target cell(s) is fundamental to an understanding of the viral persistence as well as the inflammation and demyelination observed in the chronic phase of the disease. This paper reports that a small fraction of brain macrophages grown in vitro can be efficiently infected with Theiler's virus without significant cytolytic effect. Viral replication as well as continuous production of infectivity were observed in these cultures.

The human poliovirus receptor consists of three extracellular immunoglobulinlike domains, a transmembrane domain, and an intracytoplasmic domain. The amino-terminal variable-type domain (V domain) of the human poliovirus receptor is necessary and sufficient for its function as a viral receptor (H.-C. Selinka, A. Zibert, and E. Wimmer, Proc. Natl. Acad. Sci. USA 88:3598-3602, 1991). In this paper, data are presented showing that transfer of the putative poliovirus receptor-binding domain to a truncated receptor for the human immunodeficiency virus results in a functional receptor for poliovirus. After expression in mouse cells, this chimeric protein confers susceptibility to poliovirus. Thus, unlike human immunodeficiency virus, poliovirus can enter mouse cells by way of a truncated CD4 receptor if the specific binding domain for poliovirus is provided.

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3β and nuclear translocation of the p65 subunit of NF-κB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca 2+ concentration in the endosome. In this paper, we monitored rotavirus entry by measuring α-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca 2+ concentration on virus entry was studied by inhibiting the endosomal H + -ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-α-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca 2+ -dependent endocytosis model.

Human cytomegalovirus (HCMV), a betaherpesvirus, is pathogenic almost exclusively in immunocompromised individuals. Cellular immune response is thought to control HCMV in immunocompetent people, both in the acute phase and in the latent infection which ensues (for a review, see reference 6). Cytotoxic CD8+ T cells contribute greatly to immunological control, as revealed by the presence of cytotoxic CD8+ T cells in patients who recover from acute infections (37) and by the prevention of HCMV disease following the injection of viral matrix-specific CD8+ clones to bone marrow transplant patients (44). CD4+ T cells against HCMV proteins have also been described (4, 12, 28), and a significant response towards IE1, the major immediate-early DNA-binding protein, has been reported (3, 9). The precursor frequencies of IE1-specific CD4+ T cells are high in latently infected individuals (9), and cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) found in the supernatant of CD4+ T-cell clones specific for IE1 significantly reduce HCMV replication (10). The role of CD4+ T cells in HCMV infections was inferred from these in vitro studies and from in vivo studies showing that CD4+ T cells are required for the clearance of mouse cytomegalovirus (MCMV) in salivary glands of mice (22). Major histocompatibility complex (MHC) class II expression is a key element in the control of the immune response. It is required for the activation of T lymphocytes in the process of antigen (Ag) presentation by professional or nonprofessional antigen-presenting cells (APC) (13). Three essential transactivators of MHC class II genes have been identified: RFX5 and RFX-AP are part of the RFX complex that binds to the X box of all MHC class II promoters, while the class II transactivator (CIITA) is itself tightly regulated, and this transactivator functions as the master controller of MHC class II expression, probably in all physiological conditions (30, 42). Both constitutive and inducible expressions of MHC class II genes are indeed dependent on CIITA, and the CIITA gene is itself controlled by distinct alternative promoters (33). While CIITA promoters I and III are specific for constitutive expression in dendritic cells and B lymphocytes, respectively, induction of CIITA, and hence of MHC class II, by IFN-γ is mediated by promoter IV. Activation by IFN-γ takes place via a cascade of events which involves tyrosine phosphorylation of STAT1 by JAK1 and JAK2 kinases, followed by nuclear translocation and binding of STAT1 to a GAS sequence within the promoter of the target gene (reviewed in reference 8). In the case of the activation of CIITA promoter IV by IFN-γ, cooperation between three distinct and essential transacting factors, STAT1 and USF-1 (binding cooperatively to a GAS-E box motif) and IRF-1, has recently been described in detail (34). This also explains why JAK1 and STAT1 mutants lack inducibility of MHC class II by IFN-γ (31). The detailed dissection of these multiple steps of the cascade, from IFN-γ activation to expression of MHC class II molecules (33, 34), now allows the assignment of various types of inhibition to specific steps within this cascade. APC capacity is triggered by IFN-γ treatment in many cell types, due to the induction of expression of HLA class II, the invariant chain, and HLA-DM which catalyzes the release of invariant chain-derived Clip peptide from HLA class II molecules and allows the binding of processed peptides (36). MHC class II molecules are known to present peptides derived from extracellular, exogenous Ag to CD4+ T cells (36). However, it appears that presentation of intracellular endogenous, especially viral, Ags is common (14, 23). This suggests cognate interaction between CD4+ T cell and the infected MHC class II-positive APC. Immunological controls of cytomegaloviruses are counterbalanced by escape mechanisms from class I-mediated recognition by CD8+ T cells, as recently described (46). Several genes localized in the unique short (US) region of the HCMV genome are responsible for down regulating HLA class I expression at multiple levels through retrotranslocation of HLA class I heavy chain to the cytosol (US2 [19, 47] and US11 [21, 45]), retention in the endoplasmic reticulum (US3 [1, 20]) and inhibition of TAP translocation of peptides (US6 [2, 17, 26]). The CD4+ compartment of cellular immunity has been suspected to be down regulated since the observation by Sedmak et al. (38) that HCMV inhibits HLA class II expression induced by IFN-γ. A recent report by Miller et al. (32) has described a mechanism of JAK1 proteolysis by HCMV to explain the inhibition of IFN-γ-mediated induction of HLA class II. In mice, Heise et al. (15) have reported that MCMV down regulates IFN-γ-induced expression of MHC class II-related molecules through the inhibition of their transcription. The mechanism, which occurs downstream of STAT1 activation, has yet to be determined. In this paper, we made use of an astrocytoma cell line, U373 MG, which is fully permissive to HCMV. This cell line can also be induced to express HLA-DR by treatment with IFN-γ and to subsequently present Ag to HCMV IE1-specific CD4+ T cells (11). We show here that HCMV infection prevents induction of HLA-DR expression by IFN-γ in U373 MG cells. This inhibition occurs downstream of the activation of STAT1, whose phosphorylation and nuclear translocation are not inhibited by HCMV. CIITA transcription is strongly repressed by cotreatment of cells with IFN-γ plus HCMV. Logically, this strong reduction of MHC class II expression by HCMV results in the absence of IE1 presentation and recognition by CD4+ T-cell clones. Transfection of the U373 MG cells by CIITA, however, can restore both HLA-DR expression and the ability to present endogenously produced IE1 to CD4+ T cells, even following HCMV infection. These results indicate that, in IFN-γ-treated cells, down regulation of MHC class II expression by HCMV results from a repression of CIITA induction. The modulation of MHC class II-dependent IE1 recognition by viral infection, via an effect on CIITA expression, can thus allow HCMV-infected cells to escape from the CD4+ T-cell response.

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the orderMononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr’s functions may take place at the NPC. The accessory gene vpr is present in all viruses that belong to the five phylogenetic lineages of primate lentiviruses. The human immunodeficiency virus type 1 (HIV-1) Vpr protein is well conserved in terms of primary sequence and is approximately 96 amino acids in length. To date, a number of functions have been ascribed to HIV-1 Vpr (18). First, Vpr has been shown to act early in viral infection as a facilitator of postentry nucleoprotein complex (often referred to as the preintegration complex [PIC]) nuclear import (30, 40). This activ

RmI is a circular DNA molecule that consists of a complete polyomavirus genome with an insertion (Ins) of mouse cellular DNA. This polyomavirus genome carries a mutation which renders its replication, but not its transforming ability, temperature sensitive. Ins contains both unique and repetitive cellular DNA sequences. We transfected RmI into rat cells at the permissive and nonpermissive temperatures for replication and isolated clones that had integrated RmI in their genomes. In this paper, we describe detailed mapping of the integrated RmI sequences present in 37 different cell clones. Our results indicated that transfection at the permissive temperature resulted in a random integration pattern, whereas transfection at the nonpermissive temperature resulted in a nonrandom integration pattern. The nonrandom insertions had a preferential length and preferential endpoints. We argue from these results that the nonrandom integration pattern is related to the presence of Ins and that the switch between nonrandom integration and random integration reflects a modification of the integrating substrate. When both are active, the random mechanism dominates the nonrandom mechanism.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.

The oncogenic avian retrovirus OK10 has the genetic structure gag-delta pol-myc-delta-env. The myc sequence is transduced from a cellular gene, proto-myc, while gag, pol, and env are essential retrovirus genes. By analogy with other directly oncogenic retroviruses, the specific myc sequence of OK10 is thought to be essential for transforming function. However, unlike the specific sequences of all other transforming retroviruses that encode unique transforming proteins, the myc sequence of OK10 encodes two potential transforming proteins, p58 and p200. p200 is translated from the gag-delta pol-myc region of genomic RNA, while p58 is thought to be translated from the gag leader and the open reading frame of myc via a subgenomic mRNA. In this paper, we ask whether both myc genes of OK10 are autonomous transforming genes. By differentially inactivating the p200 myc gene of OK10 provirus in vitro and analyzing transforming function in quail embryo cells, it was found that mutants expressing only p58 transformed like wild-type OK10. Further, it was shown that p58 with and without the gag leader had transforming function and that p58 of wild-type OK10 is initiated from the gag leader. Mutants expressing only p200 were also transforming but less efficiently than mutants that express only p58. A mutant OK10 virus in which the native frameshift of retroviruses between gag and pol was deleted expressed a shortened p200 (delta p200). Although this virus expressed more delta p200 than wild-type OK10 did, it transformed cells less efficiently. It follows that OK10 expresses two autonomous transforming genes, which is unique among retroviruses with onc genes.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.