Cell-cell transmission allows human T-lymphotropic virus 1 to circumvent tetherin restriction.

Tetherin is part of the cellular innate immunity and impedes cell-free transmission of viruses that bud from the plasma membrane by retaining them on the cell surface. Some viruses have evolved activities in different proteins such as Vpu (HIV-1), K-protein (KSHV), Nef (SIV) or Env (HIV-2) to downregulate tetherin and overcome its restriction. We found that chronically HTLV-1 infected T-cell lines express eightfold more tetherin than uninfected transformed T-cell lines suggesting that tetherin expression is not inhibited by the virus. We observed that even small amounts of exogenous tetherin caused the retention of HTLV-1 on the cell surface and severely reduced cell-free infectivity of HTLV-1, but that cell-cell transmission, which is more relevant for HTLV-1, was significantly less decreased. However, knock-down of tetherin expresssion resulted in a slight increase in cell-cell infection indicating that the protein does not enhance this route of transmission.

Human T-cell leukemia virus type 1 integration target sites in the human genome: comparison with those of other retroviruses.

Retroviral integration into the host genome is not entirely random, and integration site preferences vary among different retroviruses. Human immunodeficiency virus (HIV) prefers to integrate within active genes, whereas murine leukemia virus (MLV) prefers to integrate near transcription start sites and CpG islands. On the other hand, integration of avian sarcoma-leukosis virus (ASLV) shows little preference either for genes, transcription start sites, or CpG islands. While host cellular factors play important roles in target site selection, the viral integrase is probably the major viral determinant. It is reasonable to hypothesize that retroviruses with similar integrases have similar preferences for target site selection. Although integration profiles are well defined for members of the lentivirus, spumaretrovirus, alpharetrovirus, and gammaretrovirus genera, no members of the deltaretroviruses, for example, human T-cell leukemia virus type 1 (HTLV-1), have been evaluated. We have mapped 541 HTLV-1 integration sites in human HeLa cells and show that HTLV-1, like ASLV, does not specifically target transcription units and transcription start sites. Comparing the integration sites of HTLV-1 with those of ASLV, HIV, simian immunodeficiency virus, MLV, and foamy virus, we show that global and local integration site preferences correlate with the sequence/structure of virus-encoded integrases, supporting the idea that integrase is the major determinant of retroviral integration site selection. Our results suggest that the global integration profiles of other retroviruses could be predicted from phylogenetic comparisons of the integrase proteins. Our results show that retroviruses that engender different insertional mutagenesis risks can have similar integration profiles.

Resistance of human T cell leukemia virus type 1 to APOBEC3G restriction is mediated by elements in nucleocapsid.

Human T cell leukemia virus type 1 (HTLV-1) has evolved a remarkable strategy to thwart the antiviral effects of the cellular cytidine deaminase APOBEC3G (hA3G). HTLV-1 infects T lymphocytes in vivo, where, like HIV-1, it is likely to encounter hA3G. HIV-1 counteracts the innate antiviral activity of hA3G by producing an accessory protein, Vif, which hastens the degradation of hA3G. In contrast, HTLV-1 does not encode a Vif homologue; instead, HTLV-1 has evolved a cis-acting mechanism to prevent hA3G restriction. We demonstrate here that a peptide motif in the C terminus of the HTLV-1 nucleocapsid (NC) domain inhibits hA3G packaging into nascent virions. Mutation of amino acids within this region resulted in increased levels of hA3G incorporation into virions and increased susceptibility to hA3G restriction. Elements within the C-terminal extension of the NC domain are highly conserved among the primate T cell leukemia viruses, but this extension is absent in all other retroviral NC proteins.

Phenotypic and genotypic comparisons of human T-cell leukemia virus type 1 reverse transcriptases from infected T-cell lines and patient samples.

It is well established that cell-free infection with human T-cell leukemia virus type 1 (HTLV-1) is less efficient than that with other retroviruses, though the specific infectivities of only a limited number of HTLV-1 isolates have been quantified. Earlier work indicated that a post-entry step in the infectious cycle accounted for the poor cell-free infectivity of HTLV-1. To determine whether variations in the pol gene sequence correlated with virus infectivity, we sequenced and phenotypically tested pol genes from a variety of HTLV-1 isolates derived from primary sources, transformed cell lines, and molecular clones. The pol genes and deduced amino acid sequences from 23 proviruses were sequenced and compared with 14 previously published sequences, revealing a limited number of amino acid variations among isolates. The variations appeared to be randomly dispersed among primary isolates and proviruses from cell lines and molecular clones. In addition, there was no correlation between reverse transcriptase sequence and the disease phenotype of the original source of the virus isolate. HTLV-1 pol gene fragments encoding reverse transcriptase were amplified from a variety of isolates and were subcloned into HTLV-1 vectors for both single-cycle infection and spreading-infection assays. Vectors carrying pol genes that matched the consensus sequence had the highest titers, and those with the largest number of variations from the consensus had the lowest titers. The molecular clone from CS-1 cells had four amino acid differences from the consensus sequence and yielded infectious titers that were approximately eight times lower than those of vectors encoding a consensus reverse transcriptase.

Synthesis, processing, and composition of the virion-associated HTLV-1 reverse transcriptase.

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Infectious transmission and replication of human T-cell leukemia virus type 1.

Retrovirus infection proceeds by attachment of the envelope glycoprotein to a cell surface receptor, followed by fusion of the viral and cellular membranes. Once in the cell, the viral enzymes and structural proteins form a replication complex that converts the single-stranded viral genomic RNA into a double-stranded DNA, which is then integrated into the host cell chromosome. For HTLV-1, these events are not well characterized. We have developed cell culture systems, infectious molecular clones, and viral vectors that can be used to characterize the mechanisms of HTLV-1 infection and replication. Infection with cell-free HTLV-1 virions is orders of magnitude less efficient compared with other retroviruses. This inefficiency is the result of a block in the replication process after the virion is bound to the cell surface. We are determining whether this block is conferred by the viral replication enzymes, results from the actions of cellular restriction factors, reflects the need for cell-cell contact, or is caused by a combination of these factors.

cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing.

Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping.

HTLV-1-encoded p30II is a post-transcriptional negative regulator of viral replication.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.

Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs.

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex mRNA compared to pX-tax-orfII and pX-rex-orfI mRNAs, but did not affect exon skipping. Ectopic expression of hnRNP A1 did not affect pX splice site utilization, but increased exon skipping, as the level of pX-p21rex mRNA was increased by almost 10-fold.

Activation of interleukin-13 expression in T cells from HTLV-1-infected individuals and in chronically infected cell lines.

Human T-cell leukemia virus type 1 (HTLV-1) infection profoundly alters T-cell gene expression, and the dysregulated synthesis of cytokines could influence the course and pathologic consequences of infection. In the process of screening T-cell lines for T helper 1 (Th1) and Th2 cytokine mRNAs, we observed that interleukin-13 (IL-13) mRNA was highly expressed in HTLV-1-infected, IL-2-dependent T-cell lines. IL-9 and interferon gamma (IFN-gamma) mRNAs were also expressed at high levels in chronically infected cell lines. IL-5 mRNA was detected in 60% of the HTLV-1-infected cell lines, but mRNAs for IL-4, IL-10, IL-2, and IL-15 were either below detection limits or did not correlate with HTLV-1 infection. Transcriptional activation of the IL-13 promoter by the HTLV-1 Tax trans-regulatory protein was demonstrated in Jurkat T cells transiently transfected with an IL-13 promoter-reporter plasmid. The clinical relevance of these observations was demonstrated by immunofluorescent staining and flow cytometry of lymphocytes obtained from HTLV-1-infected patients. These studies revealed that IL-13 production was directly related to the level of Tax expression in the infected CD4+ T cells soon after in vitro culture. As IL-13 plays key roles in tumor immunosurveillance, asthma, and central nervous system inflammation, it may contribute to the pathophysiology of HTLV-1-associated diseases.