Discovery and characterization of auxiliary proteins encoded by type 3 simian T-cell lymphotropic viruses.

UNLABELLED: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Hyperediting of human T-cell leukemia virus type 2 and simian T-cell leukemia virus type 3 by the dsRNA adenosine deaminase ADAR-1.

RNA editing mediated by adenosine deaminases acting on RNA (ADARs) converts adenosine (A) to inosine (I) residues in dsRNA templates. While ADAR-1-mediated editing was essentially described for RNA viruses, the present work addresses the issue for two δ-retroviruses, human T-cell leukemia virus type 2 and simian T-cell leukemia virus type 3 (HTLV-2 and STLV-3). We examined whether ADAR-1 could edit HTLV-2 and STLV-3 virus genomes in cell culture and in vivo. Using a highly sensitive PCR-based method, referred to as 3DI-PCR, we showed that ADAR-1 could hypermutate adenosine residues in HTLV-2. STLV-3 hypermutation was obtained without using 3DI-PCR, suggesting a higher mutation frequency for this virus. Detailed analysis of the dinucleotide editing context showed preferences for 5' ArA and 5' UrA. In conclusion, the present observations demonstrate that ADAR-1 massively edits HTLV-2 and STLV-3 retroviruses in vitro, but probably remains a rare phenomenon in vivo.

New STLV-3 strains and a divergent SIVmus strain identified in non-human primate bushmeat in Gabon.

BACKGROUND: Human retroviral infections such as Human Immunodeficiency Virus (HIV) or Human T-cell Lymphotropic Virus (HTLV) are the result of simian zoonotic transmissions through handling and butchering of Non-Human Primates (NHP) or by close contact with pet animals. Recent studies on retroviral infections in NHP bushmeat allowed for the identification of numerous Simian Immunodeficiency Viruses (SIV) and Simian T-cell Lymphotropic Viruses (STLV) to which humans are exposed. Nevertheless, today, data on simian retroviruses at the primate/hunter interface remain scarce. We conducted a pilot study on 63 blood and/or tissues samples derived from NHP bushmeat seized by the competent authorities in different locations across the country. RESULTS: SIV and STLV were detected by antibodies to HIV and HTLV antigens, and PCRs were performed on samples with an HIV or/and HTLV-like or indeterminate profile. Fourteen percent of the samples cross-reacted with HIV antigens and 44% with HTLV antigens. We reported STLV-1 infections in five of the seven species tested. STLV-3 infections, including a new STLV-3 subtype, STLV-1 and -3 co-infections, and triple SIV, STLV-1, STLV-3 infections were observed in red-capped mangabeys (C.torquatus). We confirmed SIV infections by PCR and sequence analyses in mandrills, red-capped mangabeys and showed that mustached monkeys in Gabon are infected with a new SIV strain basal to the SIVgsn/mus/mon lineage that did not fall into the previously described SIVmus lineages reported from the corresponding species in Cameroon. The same monkey (sub)species can thus be carrier of, at least, three distinct SIVs. Overall, the minimal prevalence observed for both STLV and SIV natural infections were 26.9% and 11.1% respectively. CONCLUSIONS: Overall, these data, obtained from a restricted sampling, highlight the need for further studies on simian retroviruses in sub-Saharan Africa to better understand their evolutionary history and to document SIV strains to which humans are exposed. We also show that within one species, a high genetic diversity may exist for SIVs and STLVs and observe a high genetic diversity in the SIVgsn/mon/mus lineage, ancestor of HIV-1/SIVcpz/SIVgor.

HTLV-3/STLV-3 and HTLV-4 viruses: discovery, epidemiology, serology and molecular aspects.

Human T cell leukemia/lymphoma virus Type 1 and 2 (HTLV-1 and HTLV-2), together with their simian counterparts (STLV-1, STLV-2), belong to the Primate T lymphotropic viruses group (PTLV). The high percentage of homologies between HTLV-1 and STLV-1 strains, led to the demonstration that most HTLV-1 subtypes arose from interspecies transmission between monkeys and humans. STLV-3 viruses belong to the third PTLV type and are equally divergent from both HTLV-1 and HTLV-2. They are endemic in several monkey species that live in West, Central and East Africa. In 2005, we, and others reported the discovery of the human homolog (HTLV-3) of STLV-3 in two asymptomatic inhabitants from South Cameroon whose sera exhibited HTLV indeterminate serologies. More recently, two other cases of HTLV-3 infection in persons living in Cameroon were reported suggesting that this virus is not extremely rare in the human population living in Central Africa. Together with STLV-3, these human viral strains belong to the PTLV-3 group. A fourth HTLV type (HTLV-4) was also discovered in the same geographical area. The overall PTLV-3 and PTLV-4 genomic organization is similar to that of HTLV-1 and HTLV-2 with the exception of their long terminal repeats (LTRs) that contain only two 21 bp repeats. As in HTLV-1, HTLV-3 Tax contains a PDZ binding motif while HTLV-4 does not. An antisense transcript was also described in HTLV-3 transfected cells. PTLV-3 molecular clones are now available and will allow scientists to study the viral cycle, the tropism and the possible pathogenicity in vivo. Current studies are also aimed at determining the prevalence, distribution, and modes of transmission of these viruses, as well as their possible association with human diseases. Here we will review the characteristics of these new simian and human retroviruses, whose discovery has opened new avenues of research in the retrovirology field.

Genetic characterization of the complete genome of a highly divergent simian T-lymphotropic virus (STLV) type 3 from a wild Cercopithecus mona monkey.

BACKGROUND: The recent discoveries of novel human T-lymphotropic virus type 3 (HTLV-3) and highly divergent simian T-lymphotropic virus type 3 (STLV-3) subtype D viruses from two different monkey species in southern Cameroon suggest that the diversity and cross-species transmission of these retroviruses are much greater than currently appreciated. RESULTS: We describe here the first full-length sequence of a highly divergent STLV-3d(Cmo8699AB) virus obtained by PCR-based genome walking using DNA from two dried blood spots (DBS) collected from a wild-caught Cercopithecus mona monkey. The genome of STLV-3d(Cmo8699AB) is 8913-bp long and shares only 77% identity to other PTLV-3s. Phylogenetic analyses using Bayesian and maximum likelihood inference clearly show that this highly divergent virus forms an independent lineage with high posterior probability and bootstrap support within the diversity of PTLV-3. Molecular dating of concatenated gag-pol-env-tax sequences inferred a divergence date of about 115,117 years ago for STLV-3d(Cmo8699AB) indicating an ancient origin for this newly identified lineage. Major structural, enzymatic, and regulatory gene regions of STLV-3d(Cmo8699AB) are intact and suggest viral replication and a predicted pathogenic potential comparable to other PTLV-3s. CONCLUSION: When taken together, the inferred ancient origin of STLV-3d(Cmo8699AB), the presence of this highly divergent virus in two primate species from the same geographical region, and the ease with which STLVs can be transmitted across species boundaries all suggest that STLV-3d may be more prevalent and widespread. Given the high human exposure to nonhuman primates in this region and the unknown pathogenicity of this divergent PTLV-3, increased surveillance and expanded prevention activities are necessary. Our ability to obtain the complete viral genome from DBS also highlights further the utility of this method for molecular-based epidemiologic studies.