Species-specific inhibition of foamy viruses from South American monkeys by New World Monkey TRIM5{alpha} proteins.

Foamy virus evolution closely parallels that of the host species, indicating virus-host coadaptation. We studied simian foamy viruses (SFVs) from common marmosets, spider monkeys, and squirrel monkeys, New World monkey (NWM) species that share geographic ranges. The TRIM5alpha protein from each of these NWM species inhibited the replication of at least one of the SFVs associated with the other two species but did not affect the replication of its own SFV. Thus, TRIM5alpha has potentially shaped the evolution of SFVs in NWM hosts. Conversely, SFVs may have influenced the evolution of TRIM5 variants in New World primates.

Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state.

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus-cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high-potential-energy viral entry machines that are normally activated by receptor binding.

A human TRIM5alpha B30.2/SPRY domain mutant gains the ability to restrict and prematurely uncoat B-tropic murine leukemia virus.

Human TRIM5alpha restricts N-tropic murine leukemia virus (N-MLV) but not B-tropic MLV (B-MLV) infection. Here we study B30.2/SPRY domain mutants of human TRIM5alpha that acquire the ability to inhibit B-MLV infection prior to reverse transcription without losing the ability to restrict N-MLV infection. Remarkably, these mutants gain the ability to decrease the amount of particulate B-MLV capsids in the cytosol of infected cells. In addition, these mutants gain the ability to restrict SIV(mac) and HIV-2 infection. B-MLV and SIV(mac) infections were blocked by the mutant TRIM5alpha proteins prior to reverse transcription. Thus, the range of retroviruses restricted by human TRIM5alpha can be increased by changes in the B30.2/SPRY domain, which also result in the ability to cause premature uncoating of the restricted retroviral capsid.

Comparison of LTR enhancer elements in sheep beta retroviruses: insights into the basis for tissue-specific expression.

Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor virus (ENTV), and endogenous sheep retroviruses (ESRVs) are highly related sheep betaretroviruses that display different expression profiles in vivo. JSRV and ENTV are expressed in lungs and nasal adenocarcinomas, respectively, while ESRVs are primarily expressed in the reproductive tract of ewes. Evidence suggests that the cell tropism of JSRV, ENTV, and ESRVs is due to the transcriptional specificity of the LTRs. We have previously found several enhancer elements in the JSRV LTR that are important for lung-specific expression, including binding sites for the lung-specific transcription factor HNF-3beta, as well as binding sites for the ubiquitously expressed transcription factors C/EBP and NF-I. In this study, we have aligned the U3 regions of JSRV, ENTV, and several ESRVs in order to compare the transcriptional enhancer elements of JSRV that are conserved or absent in ESRV and ENTV. All three JSRV U3 sequences examined contain two conserved HNF-3 binding sites, while the ENTV and ESRV U3 regions are not predicted to bind this transcription factor. In addition, the C/EBP binding site is interrupted in the ESRV LTRs, but conserved in the ENTV LTRs. Some enhancer elements are conserved between JSRV and ENTV, but a reporter vector carrying the ENTV-1 LTR showed less activity than a JSRV LTR-driven reporter vector in a lung epithelial cell line. These studies support the importance of LTR enhancer elements in the respective tissue specificities of these exogenous and endogenous betaretroviruses.

In vivo and in vitro analysis of factor binding sites in Jaagsiekte sheep retrovirus long terminal repeat enhancer sequences: roles of HNF-3, NF-I, and C/EBP for activity in lung epithelial cells.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep that arises from type II pneumocytes and Clara cells of the lung epithelium. Studies of the tropism of this virus have been hindered by the lack of an efficient system for viral replication in tissue culture. To map regulatory regions important for transcriptional activation, an in vivo footprinting method that couples dimethyl sulfate treatment and ligation-mediated PCR was performed in murine type II pneumocyte-derived MLE-15 cells infected with a chimeric Moloney murine leukemia virus driven by the JSRV enhancers (DeltaMo+JS Mo-MuLV). In vivo footprints were found in the JSRV enhancers in two regions previously shown to be important for JSRV long terminal repeat (LTR) activity: a binding site for the lung-specific transcription factor HNF-3beta and an E-box element in the distal enhancer adjacent to an NF-kappaB-like binding site. In addition, in vivo footprints were detected in two downstream motifs likely to bind C/EBP and NF-I. Mutational analysis of a JSRV LTR reporter construct (pJS21luc) revealed that the C/EBP binding site is critical for LTR activity, while the putative NF-I binding element is less important; elimination of these sites resulted in 70% and 40% drops in LTR activity, respectively. Electrophoretic mobility shift assays using nuclear extracts from MLE-15 murine Clara cell-derived mtCC1-2 cells with probes corresponding to the NF-I or C/EBP sites revealed several complexes. Antiserum directed against NF-IA, C/EBPalpha, or C/EBPbeta supershifted the corresponding protein-DNA complexes, indicating that these isoforms, which are also important for the expression of several cellular lung-specific genes, may be important for JSRV expression in lung epithelial cells.

Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1.

In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5alpha with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain.

A Moloney murine leukemia virus driven by the Jaagsiekte sheep retrovirus enhancers shows enhanced specificity for infectivity in lung epithelial cells.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer in sheep. One of the unique features of this virus is that in infected animals, the only tissues that show expression of the virus are the tumor cells in the lung. We previously showed that the JSRV long terminal repeat (LTR) is preferentially active in murine lung epithelial cell lines (MLE-15 and mtCC1-2). To further explore the tissue specificity, we inserted the JSRV enhancer sequences from the U3 region of the LTR into a Moloney murine leukemia virus (M-MuLV) LTR lacking its own enhancer sequences, to give the chimeric LTR DeltaMo + JS. Transient transfection assays indicated that the DeltaMo + JS LTR is > 5-fold more active in lung epithelial cell lines than in non-lung lines, compared to the wild-type M-MuLV LTR. This was due to preferential activity of the JSRV enhancers in lung epithelial cells. Moreover, M-MuLV driven by the DeltaMo + JS LTR was > 3 logs more infectious in MLE-15 cells compared to non-lung cell lines. This chimeric virus may facilitate investigations of the tissue-specificity of JSRV.

HNF-3beta is a critical factor for the expression of the Jaagsiekte sheep retrovirus long terminal repeat in type II pneumocytes but not in Clara cells.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a sheep lung cancer that resembles human lung adenocarcinoma or bronchioloaveolar carcinoma (BAC). JSRV is the only retrovirus that shows lung tropism and induces pulmonary carcinoma. Several lines of evidence suggest that the lung tropism for JSRV is mainly determined by the viral long terminal repeats (LTR). In a previous study, we showed that HNF-3alpha and -3beta were able to transactivate the JSRV LTR when cotransfected into 3T3 cells. The JSRV LTR contains two putative HNF-3 binding sites; to investigate the contribution of each HNF-3 binding site to transcription, we generated reporter constructs with deletions or nucleotide substitutions in one or both of the putative HNF-3 binding sites. In murine MLE-15 cells (derived from type II pneumocytes), mutations within the upstream site (minus sign147 to minus sign128 bp) resulted in a 72% reduction of the LTR activity, while mutation of the downstream site had little effect. In contrast, transactivation of the JSRV LTR was greatly reduced in 3T3 cells cotransfected with an HNF-3alpha or -3beta expression plasmid when the downstream site was eliminated. Electrophoretic mobility shift assays (EMSA) revealed that nuclear extracts from MLE-15 cells, but not 3T3 cells, were able to form a retarded complex with oligonucleotides encompassing either the upstream or the downstream sites. Anti-HNF-3beta antiserum, but not anti-HNF-3alpha antiserum, supershifted both protein-DNA complexes. These results indicate that the JSRV LTR is activated by the lung-specific transcription factor HNF-3beta and that the upstream HNF-3 binding site is essential for expression in MLE-15 cells. In contrast, transactivation by HNF-3beta in 3T3 cells is mediated through the downstream HNF-3 site. On the other hand, JSRV LTR expression in a mouse lung Clara cell-derived line (mtCC1-2) did not appear to be strongly dependent on either HNF-3 binding site. These results support the notion that JSRV lung tropism is determined by the transcriptional specificity of the JSRV LTR, which is governed by interactions with lung-specific transcription factors.