TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner.

Sindbis virus (SINV) produces the small membrane protein TF from the 6K gene via a (-1) programmed ribosomal frameshifting. While several groups have shown that TF-deficient virus exhibits reduced virulence, the mechanism(s) by which this occurs remain unknown. Here, we demonstrate a role for TF in antagonizing the host interferon response. Using wild-type and type 1 interferon receptor-deficient mice and primary cells derived from these animals, we show that TF controls the induction of the host interferon response at early times during infection. Loss of TF production leads to elevated interferon and a concurrent reduction in viral loads with a loss of pathogenicity. Palmitoylation of TF has been shown to be important for particle assembly and morphology. We find that palmitoylation of TF also contributes to the ability of TF to antagonize host interferon responses as dysregulated palmitoylation of TF reduces virulence in a manner similar to loss of TF.

Mechanisms of Filovirus Entry.

Filovirus entry into cells is complex, perhaps as complex as any viral entry mechanism identified to date. However, over the past 10 years, the important events required for filoviruses to enter into the endosomal compartment and fuse with vesicular membranes have been elucidated (Fig. 1). Here, we highlight the important steps that are required for productive entry of filoviruses into mammalian cells.

Development and characterization of an equine infectious anemia virus Env-pseudotyped reporter virus.

We developed a replication-defective reporter virus pseudotyped with the envelope glycoprotein of equine infectious anemia virus (EIAV). The in vitro host range and neutralization phenotype of EIAV Env-pseudotyped virus were similar to those of replication-competent virus. An EIAV Env pseudovirus will improve antigenic characterization of viral variants and evaluation of lentivirus vaccines.

DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus.

In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR. Electrophoretic mobility shift assays were carried out to determine which transcription factor binding sites within the LTR enhancer region were bound by DH82 nuclear extracts. It was found that five different motifs were occupied. The ets motifs that are bound by PU.1 in primary macrophage nuclear extracts specifically interacted with DH82 nuclear extracts. In addition, the PEA-2, Lvb and Oct motifs that are occupied by fibroblast nuclear extracts were also bound by DH82 nuclear extracts. Finally, the methylation-dependent binding protein (MDBP) site that is bound by all nuclear extracts investigated to date demonstrated specific interactions with DH82 nuclear extracts. The observation that both macrophage-specific and fibroblast-specific motifs were utilized by DH82 nuclear extracts suggested that both macrophage-adapted and fibroblast-adapted EIAV could replicate in DH82 cells. Indeed, infectivity studies demonstrated that strains of virus that exclusively replicate in macrophages can replicate in DH82 cells and fibroblast-adapted strains of virus can also replicate in these cells. Finally, these cells could be transfected readily with the EIAV molecular clone, pSPeiav19-2, and virus spread was detected within the culture. In conclusion, this study has identified a useful cell line that should facilitate the study of EIAV expression and replication.

Cell specificity of the transcription-factor repertoire used by a lentivirus: motifs important for expression of equine infectious anemia virus in nonmonocytic cells.

The equine infectious anemia virus (EIAV) long-terminal repeat (LTR) has been identified as highly variable, both in infected horses and in cell culture. This nucleotide hypervariation is localized to the LTR enhancer region. The EIAV LTR has been implicated in controlling both the cell tropism and virulence of the virus and it is postulated that the enhancer-region hypervariation may be responsible for the LTR effects. Our previous studies have demonstrated that the presence of DNA motifs bound by the ets transcription-factor family member PU.1 are critically important for EIAV expression in equine macrophages. Here we identify and characterize the EIAV LTR enhancer motifs PEA-2, Lvb, Oct, and CRE, that bind to fibroblast nuclear extracts. Three of these four motifs, PEA-2, Oct, and CRE, were determined to be important for expression of the LTR in a fibroblast cell line that supports productive infection of EIAV. These motifs that are important for expression of the LTR in fibroblasts were found to be interdigitated between the PU.1 sites. We hypothesize that the combination of motif interdigitation and cell-specific usage of these motifs may be responsible for the observed EIAV LTR enhancer-region hypervariation.

Equine endothelial cells support productive infection of equine infectious anemia virus.

Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Infectivity was detected by cell supernatant reverse transcriptase positivity, EIAV antigen positivity within individual cells, and the detection of viral RNA by in situ hybridization. Virus could rapidly spread through the endothelial cultures, and the supernatants of infected cultures contained high titers of infectious virus. There was no demonstrable cell killing in infected cultures. Three of four strains of EIAV that were tested replicated in these cultures, including MA-1, a fibroblast-tropic strain, Th.1, a macrophage-tropic strain, and WSU5, a strain that is fibroblast tropic and can cause disease. Finally, upon necropsy of a WSU5-infected horse 4 years postinfection, EIAV-positive endothelial cells were detected in outgrowths of renal artery cultures. These findings identify a new cell type that is infectible with EIAV. The role of endothelial cell infection in the course of equine infectious anemia is currently unknown, but endothelial cell infection may be involved in the edema that can be associated with infection. Furthermore, the ability of EIAV to persistently infect endothelial cultures and the presence of virus in endothelial cells from a long-term carrier suggest that this cell type can serve as a reservoir for the virus during subclinical phases of infection.

Regulation of equine infectious anemia virus expression.

Equine infectious anemia virus (EIAV) is an ungulate lentivirus that is related to human immunodeficiency virus (HIV). Much of the understanding of lentiviral gene regulation comes from studies using HIV. HIV studies have provided insights into molecular regulation of EIAV expression; however, much of the regulation of EIAV expression stands in stark contrast to that of HIV. This review provides an overview of the current state of knowledge of EIAV regulation by comparing and contrasting EIAV gene regulation to HIV. The role of EIAV gene regulation is discussed in relation to EIAV pathogenesis.

Localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus.

The role of in vivo long terminal repeat (LTR) sequence variation of the lentivirus equine infectious anemia virus (EIAV) has not been explored. In this study, we investigated the heterogeneity found in the LTR sequences from seven EIAV-seropositive horses: three horses with clinical disease and four horses without any detectable signs of disease. LTR sequences were targeted in this study because the LTR U3 enhancer region of tissue culture-derived isolates has been identified as one of the few hypervariable regions of the EIAV genome. Furthermore, LTR variation may regulate EIAV expression in vivo. Both intra- and interanimal sequence variations were investigated. The intra-animal variation was low in seropositive, healthy horses (on average 0.44%). Intra-animal variation was consistently higher in clinically ill horses (0.99%), suggesting that greater numbers of quasispecies of EIAV are present when active virus replication is ongoing. Interanimal comparisons of consensus sequences generated from each horse demonstrated that the enhancer region is a hotspot of sequence variation in vivo. Thirty-seven of the 83 nucleotides that compose the U3 enhancer region were variable between the different in vivo-derived LTRs. The remainder of the LTR that was analyzed was more conserved, 8 of 195 nucleotide positions being variable. Results of electrophoretic mobility shift assays demonstrated that some nucleotide substitutions that occurred in the enhancer region eliminated or altered transcription factor binding motifs that are known to be important for EIAV LTR expression. These data suggested that the selective pressures exerted on the EIAV LTR enhancer sequences are different from those exerted on the remainder of the LTR. Our findings are consistent with the possibility that enhancer sequence hypervariability can alter expression of the virus in tissue macrophages and therefore contribute to clinical disease in infected horses.

Monocyte maturation controls expression of equine infectious anemia virus.

In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were infected with a macrophage-tropic strain of EIAV, and expression of EIAV was monitored in cells held as nonadherent monocytes and cells allowed to adhere and differentiate into macrophages. A 2- to 3-day delay in viral antigen expression was observed in the nonadherent cells. This restriction of viral expression in monocytes was supported by nuclear run-on studies demonstrating that on day 5 postinfection, the level of actively transcribed viral messages was 4.7-fold lower in monocyte cultures than in macrophage cultures. Electrophoretic mobility shift assays identified three regions of the U3 enhancer that interacted with nuclear extracts from normal equine macrophages. Each region contained the core binding motif of a family of transcription factors that includes the product of the proto-oncogene ets. Antibodies to the Ets family member PU.1 caused a supershifting of retarded bands in an electrophoretic mobility shift assay. Transfection studies of ets motif mutants demonstrated that the U3 ets sites were important in the regulation of EIAV transcription in macrophages. Interactions between the ets motif and nuclear extracts from freshly isolated, nonadherent monocytes, macrophages adherent for 1 or 2 days, or macrophages adherent for 5 days gave different patterns of retarded bands, although the binding specificities were similar with all three extracts. The different complexes formed by monocyte and macrophage nuclear extracts may explain the enhanced ability of mature macrophages to support EIAV expression.

Cellular and viral specificity of equine infectious anemia virus Tat transactivation.

Lentiviruses vary in their dependence on a functional tat gene during their viral life cycle. To begin to understand the viral and cellular parameters controlling equine infectious anemia virus (EIAV) transactivation, we investigated Tat function and Tat and LTR structural requirements necessary for successful transactivation. EIAV Tat expression was required for detection of viral antigens from a full-length provirus. The level of transactivation by EIAV Tat as measured by LTR-CAT assays correlated well with viral antigen expression. Using horse/mouse somatic cell hybrids (SCH), a single SCH line which supported EIAV transactivation was identified, indicating that the presence of specific horse chromosomes provided cellular factors required for transactivation. Transformed cell lines from several different species were also tested and found to differ in their ability to support EIAV transactivation. A canine cell line, Cf2Th, which was permissive for EIAV transactivation, and a human cell line, HeLa, which was not permissive for EIAV transactivation, were used to map regions of the LTR and Tat that were important in cell-specific transactivation. As expected, the R region of EIAV LTR was required for transactivation by EIAV Tat in all cell lines studied. Similarly, the R region of HIV LTR was necessary for transactivation by HIV Tat. However, the composition of the U3 region also influenced transactivation in a cell-specific manner. In Cf2Th cells, replacement of EIAV U3 sequences with HIV U3 sequences resulted in high basal (nontransactivated) expression, and as a result, only a twofold increase in expression was observed in the presence of EIAV Tat. Similar studies using HIV Tat demonstrated that transactivation occurred in Cf2Th cells when either EIAV or HIV U3 sequences were present in the LTR. In contrast, transactivation by either HIV or EIAV Tat in HeLa cells required the presence of HIV enhancer sequences. These findings suggested that the ability of transactivation to occur in some cell lines may involve interactions between cell-specific transcription factors and the activation domain of Tat. For transactivation in other cell lines, Tat appeared to require more ubiquitious factors that interact with both EIAV and HIV U3 sequences.

Interferon gamma induces the expression of human immunodeficiency virus in persistently infected promonocytic cells (U1) and redirects the production of virions to intracytoplasmic vacuoles in phorbol myristate acetate-differentiated U1 cells.

Interferon gamma (IFN-gamma), a lymphokine that exerts multiple immunoregulatory effects, has been found to be elevated in the plasma, cerebrospinal fluid, and lymph nodes of human immunodeficiency virus (HIV)-infected individuals and has shown variable effects on HIV replication in acutely infected cells. In the present study, we have demonstrated that IFN-gamma is a potent modulator of HIV expression in persistently infected U1 promonocytic cells in which virus production is characterized by a constitutive state of relative latency. Direct stimulation of U1 cells with IFN-gamma (10-1,000 U/ml) activated HIV expression, as measured by reverse transcriptase (RT) activity in the culture supernatant and increased levels of cell-associated viral protein and mRNAs. These effects on virus expression were not accounted for by the induction of endogenous TNF-alpha secretion, as previously described in U1 cells stimulated with phorbol myristate acetate (PMA). At the ultrastructural level, the stimulatory activity of IFN-gamma was correlated with HIV particle production in intracytoplasmic vacuoles along with the differentiation of U1 into macrophage-like cells. Furthermore, costimulation of U1 cells with IFN-gamma and PMA significantly increased the accumulation of vacuole-associated HIV concomitant with decreasing membrane-associated particles and RT activity production, as compared with cells stimulated with PMA alone. No evidence of spontaneous secretion of intracellular vacuole-associated virus was obtained by kinetic analysis of the RT activity released in the supernatants throughout the culture period unless cells were deliberately disrupted. These findings suggest that vacuole-associated virions likely represent a relatively stable intracellular reservoir of HIV, as previously described in primary macrophages infected in vitro or in infected macrophages in the brains of patients with acquired immune deficiency syndrome. The reduced levels of RT activity observed in the culture supernatants of U1 cells stimulated with PMA in the presence of IFN-gamma were not indicative of a suppressive effect of IFN-gamma on PMA-induced expression of HIV proteins and mRNAs, either directly or mediated by the release of IFN-alpha/beta. This study suggests that IFN-gamma may play an important role as an inducer of HIV expression in infected mononuclear phagocytes.

Direct and cytokine-mediated activation of protein kinase C induces human immunodeficiency virus expression in chronically infected promonocytic cells.

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.

Replication of HIV-1 in primary monocyte cultures.

A subpopulation of human monocytes began to express surface CD4 during its in vitro cultivation in the presence of recombinant M-CSF and became susceptible to infection with an isolate of HIV-1 [AD-87(M)], originally recovered from a seropositive individual by cocultivation with primary monocytes. Approximately 10% of the adherent cells synthesized CD4; a similar fraction was infectable with AD-87(M) and produced high levels of progeny particles. An interesting feature of the HIV-1 infection of primary monocytes was the detection of gp 160/120 inside but not on the surface of virus-producing cells. The implication of these results on HIV infection in vivo will be discussed.

HIV-1 infection of first-trimester and term human placental tissue: a possible mode of maternal-fetal transmission.

To understand the potential role of placental tissue in the pathogenesis of neonatal AIDS, the distribution of human immunodeficiency virus type 1 (HIV-1) receptor and the infectability of placental tissue by HIV-1 were studied. Both the mRNA and the protein for the HIV receptor (CD4) were present in fetal-derived placenta. By immunofluorescent microscopy, a number of different cell types appeared to be CD4+; positive cells were observed in the lining and stroma of the chorionic villi. Some of these CD4+ cells dual-labeled with the trophoblastic marker placental lactogen. In addition, CD4+ cells were observed within the lining of placental blood vessels. Organ cultures of first-trimester and term placentas were infectable by HIV as monitored by reverse transcriptase activity of culture supernatants and by immunofluorescent labeling of HIV antigens. One potential route of congenital HIV transmission may be direct placental infection by HIV as early as the first trimester, with subsequent transplacental spread of the virus.

Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.

Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.

Selective infection of human CD4+ cells by simian immunodeficiency virus: productive infection associated with envelope glycoprotein-induced fusion.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus share the property of tropism for CD4-bearing cells. Infection is initiated by a high-affinity interaction between CD4 and conserved domains on the viral envelope glycoprotein. In this report, we demonstrated that SIV had a restricted host range among human CD4+ cells when compared with human immunodeficiency virus type 1 or type 2. This restricted tropism was associated with the inability of the SIV envelope glycoprotein to induce membrane fusion in cells not susceptible to productive exogenous infection by SIV. We conclude that the major route of SIV entry into CD4+ cells is by envelope-mediated direct fusion with the cell and that additional envelope-cell interactions after CD4 binding are required for productive infection.

Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE.

Addition of exogenous Mg(2+) (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K(+). Addition of K(+) (10-50 millimolar) reversed both stromal acidification and K(+) efflux from the chloroplast caused by Mg(2+). These data suggested that Mg(2+) induced reversible H(+)/K(+) fluxes across the chloroplast envelope. Ca(2+) and Mn(2+) (2 millimolar) were as effective as 4 millimolar Mg(2+) in causing K(+) efflux from chloroplasts and inhibition of O(2) evolution. In contrast, 10 millimolar Ba(2+) induced only a small amount of inhibition. The lack of strong inhibition by Ba(2+) indicated that the effects of divalent cations such as Mg(2+) cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg(2+) was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O(2) evolution (43%) induced by Mg(2+).A variety of ATPase inhibitors were tested for effects on Mg(2+)-induced H(+)/K(+) fluxes. Oligomycin was the only ATPase inhibitor which specifically inhibited photosynthesis in the presence of Mg(2+) + K(+), but had little or no effect in the absence of these cations. In the presence of oligomycin, much higher concentrations (50 millimolar) of exogenous K(+) were required to reverse Mg(2+)-induced acidification and inhibition of O(2) evolution than in its absence. Oligomycin (in the absence of divalent cations) increased the inhibition of photosynthesis caused by sodium acetate, which acts by causing stromal acidification. In addition, the chloroplast envelope ATPase was inhibited partially (45%) by oligomycin. These results suggested that H(+) fluxes across the chloroplast envelope are regulated by two mechanisms: (a) an active, oligomycin-sensitive H(+) efflux and (b) a reversible, Mg(2+)-dependent, oligomycin-insensitive H(+)/K(+) exchange.