HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP.

BACKGROUND: Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. RESULTS: We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. CONCLUSIONS: The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.

The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.

Dynamic range of Nef functions in chronic HIV-1 infection.

HIV-1 Nef is required for efficient viral replication and pathogenesis. However, the extent to which Nef's functions are maintained in natural sequences during chronic infection, and their clinical relevance, remains incompletely characterized. Relative to a control Nef from HIV-1 strain SF2, HLA class I and CD4 down-regulation activities of 46 plasma RNA Nef sequences derived from unique chronic infected individuals were generally high and displayed narrow dynamic ranges, whereas Nef-mediated virion infectivity, PBMC replication and CD74 up-regulation exhibited broader dynamic ranges. 80% of patient-derived Nefs were active for at least three functions examined. Functional co-dependencies were identified, including positive correlations between CD4 down-regulation and virion infectivity, replication, and CD74 up-regulation, and between CD74 up-regulation and PBMC replication. Nef-mediated virion infectivity inversely correlated with patient CD4(±) T-cell count. Strong functional co-dependencies and the polyfunctional nature of patient-derived Nef sequences suggest a phenotypic requirement to maintain multiple Nef functions during chronic infection.

Attenuation of multiple Nef functions in HIV-1 elite controllers.

BACKGROUND: Impaired HIV-1 Gag, Pol, and Env function has been described in elite controllers (EC) who spontaneously suppress plasma viremia to < 50 RNA copies/mL; however, activity of the accessory protein Nef remains incompletely characterized. We examined the ability of 91 Nef clones, isolated from plasma of 45 EC and 46 chronic progressors (CP), to down-regulate HLA class I and CD4, up-regulate HLA class II invariant chain (CD74), enhance viral infectivity, and stimulate viral replication in PBMC. RESULTS: In general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP. Nef clones from HLA-B*57-expressing EC exhibited poorer CD4 down-regulation function compared to those from non-B*57 EC, and the number of EC-specific B*57-associated Nef polymorphisms correlated inversely with 4 of 5 Nef functions in these individuals. CONCLUSION: Results indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype.

Naturally arising HIV-1 Nef variants conferring escape from cytotoxic T lymphocytes influence viral entry co-receptor expression and susceptibility to superinfection.

HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B*3501. CTL assays corroborated that these mutations conferred escape from HLA-B(∗)3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.

The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication.

BACKGROUND/AIMS: The aim of this study is to clarify the differences of host immune responses between acute self-limited and chronic persistent hepatitis B virus (HBV) infections by quantitative and qualitative analysis of HLA-A*2402-restricted HBV-specific CD8+ T cells. METHODS: HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV were analyzed by flow cytometry using two HLA-A*2402-HBV peptide tetrameric complexes. RESULTS: High numbers of HBV-specific CD8+ T cells were detected in acute phase PBMCs from most individuals with acute HBV infection while the number of these cells was greatly reduced in recovery phase PBMCs. HBV-specific CD8+ T cells were not detected in PBMCs from individuals with chronic HBV infection except for one patient during acute exacerbation. HBV-specific CD8+ T cells were induced by in vitro peptide stimulation in PBMCs from chronic HBV carriers with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. CD28CD45RA phenotype analysis showed that HBV-specific CD8+ T cells in acute phase PBMCs predominantly express a memory T cell phenotype. CONCLUSIONS: HBV-specific memory CD8+ T cells may play a crucial role in complete clearance of HBV from patients with acute HBV hepatitis.