Specificity of interferon action in protein synthesis.

Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.     

Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells

Expression and DNA methylation of the Moloney murine leukemia virus (M-MuLV) genome were investigated in murine teratocarcinoma cells after virus infection. The newly acquired viral genome was devoid of methylation, yet its expression was repressed. The integrated viral genome in undifferentiated teratocarcinoma cells was methylated within 15 days after infection. Although 5-azacytidine decreased the level of DNA methylation, it did not activate M-MuLV in undifferentiated cells. Activation by 5-azacytidine occurred only in differentiated teratocarcinoma cells. Thus two independent mechanisms seem to regulate gene expression during the course of differentiation. The first mechanism operates in undifferentiated cells to block expression of M-MuLV and other exogeneously acquired viral genes, such as SV40 and polyoma virus, and does not depend on DNA methylation. The second mechanism relates only to differentiated cells and represses expression of genes in which DNA is methylated.     

Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70^SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70^SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.     

Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: control by a single dominant locus on chromosome 1.

Although xenotropic murine leukemia viruses cannot productively infect cells of laboratory mice, cells from various wild-derived mice can support replication of these viruses. Although the virus-sensitive wild mice generally lack all or most of the xenotropic proviral genes characteristic of inbred strains, susceptibility to exogenous infection is unrelated to inheritance of these sequences. Instead, susceptibility is controlled by a single dominant gene, designated Sxv, which maps to chromosome 1. Sxv is closely linked to, but distinct from Bxv-1, the major locus for induction of xenotropic murine leukemia viruses in laboratory mice. Genetic experiments designed to characterize Sxv show that this gene also controls sensitivity to a wild mouse virus with the interference properties of mink cell focus-forming murine leukemia viruses, and that Sxv-mediated susceptibility to xenotropic murine leukemia viruses is restricted by the mink cell focus-forming virus resistance gene Rmcf. These data, together with genetic mapping of the mink cell focus-forming virus cell surface receptor locus to this same region of chromosome 1, suggest that Sxv may encode a wild mouse variant of the mink cell focus-forming virus receptor that allows penetration by xenotropic murine leukemia viruses.     

Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.     

DNA methylation affecting the expression of murine leukemia proviruses.

The endogenous, vertically transmitted proviral DNAs of the ecotropic murine leukemia virus in AKR embryo fibroblasts were found to be hypermethylated relative to exogenous AKR murine leukemia virus proviral DNAs acquired by infection of the same cells. The hypermethylated state of the endogenous AKR murine leukemia virus proviruses in these cells correlated with the failure to express AKR murine leukemia virus and the lack of infectivity of cellular DNA. Induction of the endogenous AKR murine leukemia virus proviruses with the methylation antagonist 5-azacytidine suggested a causal connection between DNA methylation and provirus expression. Also found to be relatively hypermethylated and noninfectious were three of six Moloney murine leukemia virus proviral DNAs in an unusual clone of infected rat cells. Recombinant DNA clones which derived from a methylated, noninfectious Moloney provirus of this cell line were found to be highly active upon transfection, suggesting that a potentially active proviral genome can be rendered inactive by cellular DNA methylation. In contrast, in vitro methylation with the bacterial methylases MHpaII and MHhaI only slightly reduced the infectivity of the biologically active cloned proviral DNA. Recombinant DNA clones which derived from a second Moloney provirus of this cell line were noninfectious. An in vitro recombination method was utilized in mapping studies to show that this lack of infectivity was governed by mechanisms other than methylation.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.     

Blockade of virus infection by human CD4+ T cells via a cytokine relay network

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.     

Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses.

Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.     

Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

The murine gene Fv-1 predominantly controls the outcome of infection by murine ecotropic retroviruses. The inhibition of virus replication by the Fv-1 gene product has been determined to be at an early stage in virus replication. Mechanistically, its effect appears to be on the accumulation of unintegrated proviral DNA or its integration or both. We investigated the synthesis of unintegrated proviral DNA, using several clones of B-, N-, or NB-tropic Friend murine leukemia virus. Our results indicate that the accumulation of B-tropic proviral DNA in NIH cells may be inhibited at either the level of linear (form III) or covalently closed circular DNA (form I), depending upon the degree of restriction of the clone of virus used. We confirmed that there is an effect of the Fv-1 gene on the accumulation of form I DNA of either B- or N-tropic Friend murine leukemia virus. However, the decrease in infectious centers effected by the Fv-1 gene did not correlate quantitatively with the effect on form I proviral DNA produced by N-tropic Friend murine leukemia virus in nonpermissive cells. Lastly, we demonstrated in nonpermissively infected NIH cells that a rapidly migrating doublet of viral DNA is formed.     

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity.

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.     

Cutting edge: paradoxical roles of BST2/tetherin in promoting type I IFN response and viral infection

Bone marrow stromal Ag 2 (BST2) is a transmembrane protein that prevents virus release from infected cells. It was also reported that BST2 inhibits type I IFN production by plasmacytoid dendritic cells. To determine BST2 impact on antiviral responses in vivo, we generated BST2(-/-) mice. Following infection with a murine retrovirus, BST2(-/-) mice had slightly elevated viral loads; however, infection with other enveloped viruses revealed unexpected roles of BST2. BST2(-/-) mice showed reduced type I IFN production by plasmacytoid dendritic cells. Moreover, BST2(-/-) mice had lower viral titers in lungs following intranasal infection with vesicular stomatitis virus expressing OVA and influenza B and increased numbers of virus-specific CD8 T cells in the lungs, suggesting that BST2 may facilitate entry and/or replication of enveloped viruses and modulate priming of CD8 T cells. These findings suggest complex roles of BST2 beyond retroviral control in vivo, possibly reflecting the involvement of BST2 in endocytosis and intracellular trafficking of viruses, viral nucleic acids, and Ags.     

Susceptibility of mink (Mustera vision)-derived cells to replication by human immunodeficiency virus type 1

In vivo studies for understanding viral transmission and replication, host immune responses, and pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection would greatly benefit from the establishment of a small-animal model. In this study, we explored the potential of American mink (Mustera vison) as a susceptible host. We found that primary cells and cell lines derived from this species efficiently supported trans-activation of the HIV-1 long terminal repeat by Tat. Accordingly, the cysteine residue at position 261, which has been shown to be important for interaction of the human cyclin T1 with the HIV-1 regulatory protein Tat, is conserved in the mink homologue. No species-specific defect in Rev function could be detected in mink cells. In addition, primary splenocytes, fibroblasts, and the Mv.1.Lu cell line from American mink supported early as well as late HIV-1 gene expression following infection with vesicular stomatitis G protein-pseudotyped HIV-1 viruses, at levels comparable to those seen with permissive human cells. Furthermore, the mink Mv.1.Lu cell line stably expressing human CD4 and CCR5 receptors supported a spreading HIV-1 infection with few, if any, deficiencies compared to findings in human cell lines. This indicates the potential of HIV-1 to replicate in these cells once the blockade at the stage of virus entry has been removed. These results clearly show that cells from American mink generally pose no functional intracellular block to HIV-1 replication, and collectively they raise the possibility that this animal species could be engineered to support HIV-1 infection, providing a useful small-animal model for evaluating de novo infection by HIV-1.     

Induction of murine p30 by superinfecting herpesviruses.

The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line.     

ORF73-null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication

Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.     

Dissecting Virus Entry: Replication-Independent Analysis of Virus Binding,Internalization, and Penetration Using Minimal Complementation of β-Galactosidase

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).     

Oncolytic virotherapy: molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment.     

Production of Sindbis,influenza, and vesicular stomatitis viruses in chicken embryo and rat embryo cell suspensions

Studies were carried out on the production of Sindbis, influenza and vesicular stomatitis viruses in suspensions of chicken embryo and rat embryo cells. The yield of Sindbis virus in chicken embryo cell suspensions was independent of the multiplicity of infection over the range 0.0001 to 0.01 although reduction in multiplicity caused a delay in virus production. With influenza virus the use of higher multiplicities gave increased virus yields possibly due to the very slow production at low multiplicities. In both monolayer and suspension cultures of chicken embryo cells addition of serum or use of media richer than minimum essential medium (Eagle) had little effect on Sindbis virus production, but if the glucose were omitted the virus yield was markedly reduced. In cell suspensions, a marked reduction in virus yield occurred if infection were delayed more than 24 hr after cell preparation whereas in monolayers the delay of infection allowed cell propagation and hence a higher yield of virus. It was also shown that vesicular stomatitis virus can be produced in chicken embryo cell suspensions, and that rat embryo primary cell suspensions can be used to prepare both Sindbis and vesicular stomatitis viruses. Sindbis virus obtained from chicken embryo cell suspensions was purified by polyethylene glycol precipitation and sucrose density gradient centrifugation and shown to contain only those proteins previously identified as viral, without any contamination from chicken cell proteins. The relative ease and economics of virus production by cell suspension and monolayer methods is compared.