Origin and functional role of antisense transcription in endogenous and exogenous retroviruses.

Most proteins expressed by endogenous and exogenous retroviruses are encoded in the sense (positive) strand of the genome and are under the control of regulatory elements within the 5' long terminal repeat (LTR). A number of retroviral genomes also encode genes in the antisense (negative) strand and their expression is under the control of negative sense promoters within the 3' LTR. In the case of the Human T-cell Lymphotropic Virus 1 (HTLV-1), the antisense protein HBZ has been shown to play a critical role in the virus lifecycle and in the pathogenic process, while the function of the Human Immunodeficiency Virus 1 (HIV-1) antisense protein ASP remains unknown. However, the expression of 3' LTR-driven antisense transcripts is not always demonstrably associated with the presence of an antisense open reading frame encoding a viral protein. Moreover, even in the case of retroviruses that do express an antisense protein, such as HTLV-1 and the pandemic strains of HIV-1, the 3' LTR-driven antisense transcript shows both protein-coding and noncoding activities. Indeed, the ability to express antisense transcripts appears to be phylogenetically more widespread among endogenous and exogenous retroviruses than the presence of a functional antisense open reading frame within these transcripts. This suggests that retroviral antisense transcripts may have originated as noncoding molecules with regulatory activity that in some cases later acquired protein-coding function. Here, we will review examples of endogenous and exogenous retroviral antisense transcripts, and the ways through which they benefit viral persistence in the host.

Different Mutation Tolerance of Lentiviral (HIV-1) and Deltaretroviral (BLV and HTLV) Protease Precursors.

The bovine leukemia virus (BLV) and the human T-lymphothropic viruses (HTLVs) are members of the deltaretrovirus genus of Retroviridae family. An essential event of the retroviral life cycle is the processing of the polyproteins by the viral protease (PR); consequently, these enzymes became important therapeutic targets of the anti-retroviral drugs. As compared to human immunodeficiency viruses (HIVs), the deltaretroviruses have a different replication strategy, as they replicate predominantly in the DNA form, by forcing the infected cell to divide, unlike HIV-1, which replicates mainly by producing a vast number of progeny virions and by reinfection. Due to bypassing the error-prone reverse transcription step of replication, the PRs of deltaretroviruses did not undergo such extensive evolution as HIV PRs and remained more highly conserved. In this work, we studied the abilities of wild-type and modified BLV, HTLV (type 1, 2 and 3), and HIV-1 PRs (fused to an N-terminal MBP tag) for self-processing. We designed a cleavage site mutant MBP-fused BLV PR precursor as well, this recombinant enzyme was unable for self-proteolysis, the MBP fusion tag decreased its catalytic efficiency but showed an unusually low Ki for the IB-268 protease inhibitor. Our results show that the HTLV and BLV deltaretrovirus PRs exhibit lower mutation tolerance as compared to HIV-1 PR, and are less likely to retain their activity upon point mutations at various positions, indicating a higher flexibility of HIV-1 PR in tolerating mutations under selective pressure.

Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells.

Wnt5a is a ligand of the non-canonical Wnt signaling pathway involved in cell differentiation, motility, and inflammatory response. Adult T-cell leukemia/lymphoma (ATL) is one of the most aggressive T-cell malignancies caused by infection of human T-cell leukemia virus type1 (HTLV-1). Among subtypes of ATL, acute-type ATL cells are particularly resistant to current multidrug chemotherapies and show remarkably high cell-proliferative and invasive phenotypes. Here we show a dramatic increase of WNT5A gene expression in acute-type ATL cells compared with those of indolent-type ATL cells. Treatment with IWP-2 or Wnt5a-specific knockdown significantly suppressed cell growth of ATL-derived T-cell lines. We demonstrated that the overexpression of c-Myb and FoxM1 was responsible for the synergistic activation of the WNT5A promoter. Also, a WNT5A transcript variant without the exon4 (the ΔE4-WNT5A mRNA), encoding ΔC-Wnt5 (1-136aa of 380aa), is overexpressed in acute-type ATL cells. The ΔC-Wnt5a is secreted extracellularly and enhances cellular migration/invasion to a greater extent compared with wildtype (WT)-Wnt5a. Moreover, the ΔC-Wnt5a secretion was not suppressed by IWP-2, indicating that this mutant Wnt5a is secreted via a different pathway from the WT-Wnt5a. Taken together, synergistic overexpression of the ΔC-Wnt5a by c-Myb and FoxM1 may be responsible for the malignant phenotype of acute-type ATL cells.

North American Big Brown Bats (Eptesicus fuscus) Harbor an Exogenous Deltaretrovirus.

Bats are the reservoir for a large number of zoonotic viruses, including members of Coronaviridae (severe acute respiratory syndrome coronavirus [SARS-CoV] and SARS-CoV-2), Paramyxoviridae (Hendra and Nipah viruses), Rhabdoviridae (rabies virus), and Filoviridae (Ebola virus) as exemplars. Many retroviruses, such as human immunodeficiency virus, are similarly zoonotic; however, only infectious exogenous gammaretroviruses have recently been identified in bats. Here, viral metagenomic sequencing of samples from bats submitted for rabies virus testing, largely due to human exposure, identified a novel, highly divergent exogenous Deltaretrovirus from a big brown bat (Eptesicus fuscus) in South Dakota. The virus sequence, corresponding to Eptesicus fuscus deltaretrovirus (EfDRV), comprised a nearly complete coding region comprised of canonical 5'-gag-pro-pol-env-3' genes with 37% to 51% identity to human T-lymphotropic virus (HTLV), an infectious retrovirus that causes T-cell lymphoma. A putative tax gene with 27% identity to HTLV was located downstream of the pol gene along with a gene harbored in an alternative reading frame which possessed a conserved domain for an Epstein-Barr virus nuclear antigen involved in gene transactivation, suggesting a regulatory function similar to that of the deltaretrovirus rex gene. A TaqMan reverse transcriptase PCR (RT-PCR) targeting the pol gene identified 4/60 (6.7%) bats as positive for EfDRV, which, combined with a search of the E. fuscus genome that failed to identify sequences with homology to EfDRV, suggests that EfDRV is an infectious exogenous virus. As all known members of Deltaretrovirus can cause malignancies and E. fuscus is widely distributed in the Americas, often with a colonial roosting behavior in human dwellings, further studies are needed to investigate potential zoonosis.IMPORTANCE Bats host a large numbers of viruses, many of which are zoonotic. In the United States, the big brown bat (Eptesicus fuscus) is widely distributed and lives in small colonies that roost in cavities, often in human dwellings, leading to frequent human interaction. Viral metagenomic sequencing of samples from an E. fuscus bat submitted for rabies testing identified the first exogenous bat Deltaretrovirus The E. fuscus deltaretrovirus (EfDRV) genome consists of the typical deltaretrovial 5'-gag-pro-pol-env-3' genes along with genes encoding two putative transcriptional transactivator proteins distantly related to the Tax protein of human T-cell lymphotrophic virus and nuclear antigen 3B of Epstein-Barr virus. Searches of the E. fuscus genome sequence failed to identify endogenous EfDRV. RT-PCR targeting the EfDRV pol gene identified 4/60 (6.7%) bats with positive results. Together, these results suggest that EfDRV is exogenous. As all members of Deltaretrovirus are associated with T- and B-cell malignancies or neurologic disease, further studies on possible zoonosis are warranted.

The bZIP Proteins of Oncogenic Viruses.

Basic leucine zipper (bZIP) transcription factors (TFs) govern diverse cellular processes and cell fate decisions. The hallmark of the leucine zipper domain is the heptad repeat, with leucine residues at every seventh position in the domain. These leucine residues enable homo- and heterodimerization between ZIP domain α-helices, generating coiled-coil structures that stabilize interactions between adjacent DNA-binding domains and target DNA substrates. Several cancer-causing viruses encode viral bZIP TFs, including human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) and the herpesviruses Marek's disease virus (MDV), Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we provide a comprehensive review of these viral bZIP TFs and their impact on viral replication, host cell responses and cell fate.

Deltaretroviruses have circulated since at least the Paleogene and infected a broad range of mammalian species.

The Deltaretrovirus genus of retroviruses (family Retroviridae) includes the human T cell leukemia viruses and bovine leukemia virus (BLV). Relatively little is known about the biology and evolution of these viruses, because only a few species have been identified and the genomic 'fossil record' is relatively sparse. Here, we report the discovery of multiple novel endogenous retroviruses (ERVs) derived from ancestral deltaretroviruses. These sequences-two of which contain complete or near complete internal coding regions-reside in genomes of several distinct mammalian orders, including bats, carnivores, cetaceans, and insectivores. We demonstrate that two of these ERVs contain unambiguous homologs of the tax gene, indicating that complex gene regulation has ancient origins within the Deltaretrovirus genus. ERVs demonstrate that the host range of the deltaretrovirus genus is much more extensive than suggested by the relatively small number of exogenous deltaretroviruses described so far, and allow the evolutionary timeline of deltaretrovirus-mammal interaction to be more accurately calibrated.

Functional Analysis of Aberrantly Spliced Caspase8 Variants in Adult T-Cell Leukemia Cells.

The genetic and molecular alterations responsible for leukemogenesis and progression of HTLV-infected adult T-cell leukemia (ATL) have not been fully clarified. Previously, we reported that various genes are not only overexpressed but also abnormally spliced in ATL cells. Here, we identified various CASP8 transcript variants in PBMCs from a smoldering-type ATL patient, which encode aberrant truncated caspase 8 (Casp8) isoforms. Among those, we focus on the three transcript variants, CASP8L (including the first 136 bp of the intron 8 between exon 8 and exon 9), CASP8-ΔE4 (without the exon 4), and CASP8-ΔE7 (without the exon 7), because they encode isoforms, Casp8L, Casp8-ΔE4, and Casp8-ΔE7, respectively, without the C-terminal catalytic domains. In this study, we conducted in vitro characterization and functional analysis of those mutant Casp8 isoforms to clarify their changed functions compared with the wild-type (WT)-Casp8. We demonstrated that these abnormal Casp8 isoforms showed lower ability to induce apoptosis than WT-Casp8 due to their dominant-negative interactions with WT-Casp8, which impair WT-Casp8 homodimerization that is essential for induction of apoptosis. Moreover, Casp8L and Casp8-ΔE7, which have only two death-effector domains, significantly activated NFκB by forming filament-like structures, which probably function as scaffolds for the IKK complex formation. In view of increasing levels of these abnormal CASP8 transcripts in primary PBMCs from HTLV-1 carriers and patients with ATL, we propose a possibility that overexpression of those Casp8 mutants, with lower proapoptotic activities and higher NFκB-activating functions than WT-Casp8, may be one of the molecular abnormalities causing malignant transformation and growth of ATL cells. IMPLICATIONS: We describe naturally occurring CASP8 transcription variants in PBMCs from patients with ATL, which encode truncated Casp8-mutant isoforms with lower proapoptotic activities and higher NFκB-activating functions compared with WT-Casp8.

Remnants of an Ancient Deltaretrovirus in the Genomes of Horseshoe Bats (Rhinolophidae).

Endogenous retrovirus (ERV) sequences provide a rich source of information about the long-term interactions between retroviruses and their hosts. However, most ERVs are derived from a subset of retrovirus groups, while ERVs derived from certain other groups remain extremely rare. In particular, only a single ERV sequence has been identified that shows evidence of being related to an ancient Deltaretrovirus, despite the large number of vertebrate genome sequences now available. In this report, we identify a second example of an ERV sequence putatively derived from a past deltaretroviral infection, in the genomes of several species of horseshoe bats (Rhinolophidae). This sequence represents a fragment of viral genome derived from a single integration. The time of the integration was estimated to be 11-19 million years ago. This finding, together with the previously identified endogenous Deltaretrovirus in long-fingered bats (Miniopteridae), suggest a close association of bats with ancient deltaretroviruses.

Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure.IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and the relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically infected cell line are polymorphic in nature, with many particles lacking organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.

Discovery of an endogenous Deltaretrovirus in the genome of long-fingered bats (Chiroptera: Miniopteridae).

Retroviruses can create endogenous forms on infiltration into the germline cells of their hosts. These forms are then vertically transmitted and can be considered as genetic fossils of ancient viruses. All retrovirus genera, with the exception of deltaretroviruses, have had their representation identified in the host genome as a virus fossil record. Here we describe an endogenous Deltaretrovirus, identified in the germline of long-fingered bats (Miniopteridae). A single, heavily deleted copy of this retrovirus has been found in the genome of miniopterid species, but not in the genomes of the phylogenetically closest bat families, Vespertilionidae and Cistugonidae. Therefore, the endogenization occurred in a time interval between 20 and 45 million years ago. This discovery closes the last major gap in the retroviral fossil record and provides important insights into the history of deltaretroviruses in mammals.

Zoonotic Transmission of Two New Strains of Human T-lymphotropic Virus Type 4 in Hunters Bitten by a Gorilla in Central Africa.

Molecular screening of 300 at-risk people from Central Africa identified 2 human T-lymphotropic virus (HTLV)-4-infected individuals. A zoonotic origin of infection was suggested, as both individuals reported being severely bitten by a gorilla during hunting activities. One strain was highly divergent and was designated as the HTLV-4 subtype-b prototype.

A novel one-class SVM based negative data sampling method for reconstructing proteome-wide HTLV-human protein interaction networks.

Protein-protein interaction (PPI) prediction is generally treated as a problem of binary classification wherein negative data sampling is still an open problem to be addressed. The commonly used random sampling is prone to yield less representative negative data with considerable false negatives. Meanwhile rational constraints are seldom exerted on model selection to reduce the risk of false positive predictions for most of the existing computational methods. In this work, we propose a novel negative data sampling method based on one-class SVM (support vector machine, SVM) to predict proteome-wide protein interactions between HTLV retrovirus and Homo sapiens, wherein one-class SVM is used to choose reliable and representative negative data, and two-class SVM is used to yield proteome-wide outcomes as predictive feedback for rational model selection. Computational results suggest that one-class SVM is more suited to be used as negative data sampling method than two-class PPI predictor, and the predictive feedback constrained model selection helps to yield a rational predictive model that reduces the risk of false positive predictions. Some predictions have been validated by the recent literature. Lastly, gene ontology based clustering of the predicted PPI networks is conducted to provide valuable cues for the pathogenesis of HTLV retrovirus.

Computational reconstruction of proteome-wide protein interaction networks between HTLV retroviruses and Homo sapiens.

BACKGROUND: Human T-cell leukemia viruses (HTLV) tend to induce some fatal human diseases like Adult T-cell Leukemia (ATL) by targeting human T lymphocytes. To indentify the protein-protein interactions (PPI) between HTLV viruses and Homo sapiens is one of the significant approaches to reveal the underlying mechanism of HTLV infection and host defence. At present, as biological experiments are labor-intensive and expensive, the identified part of the HTLV-human PPI networks is rather small. Although recent years have witnessed much progress in computational modeling for reconstructing pathogen-host PPI networks, data scarcity and data unavailability are two major challenges to be effectively addressed. To our knowledge, no computational method for proteome-wide HTLV-human PPI networks reconstruction has been reported. RESULTS: In this work we develop Multi-instance Adaboost method to conduct homolog knowledge transfer for computationally reconstructing proteome-wide HTLV-human PPI networks. In this method, the homolog knowledge in the form of gene ontology (GO) is treated as auxiliary homolog instance to address the problems of data scarcity and data unavailability, while the potential negative knowledge transfer is automatically attenuated by AdaBoost instance reweighting. The cross validation experiments show that the homolog knowledge transfer in the form of independent homolog instances can effectively enrich the feature information and substitute for the missing GO information. Moreover, the independent tests show that the method can validate 70.3% of the recently curated interactions, significantly exceeding the 2.1% recognition rate by the HT-Y2H experiment. We have used the method to reconstruct the proteome-wide HTLV-human PPI networks and further conducted gene ontology based clustering of the predicted networks for further biomedical research. The gene ontology based clustering analysis of the predictions provides much biological insight into the pathogenesis of HTLV retroviruses. CONCLUSIONS: The Multi-instance AdaBoost method can effectively address the problems of data scarcity and data unavailability for the proteome-wide HTLV-human PPI interaction networks reconstruction. The gene ontology based clustering analysis of the predictions reveals some important signaling pathways and biological modules that HTLV retroviruses are likely to target.

Human T-cell leukemia virus type 3 (HTLV-3) and HTLV-4 antisense-transcript-encoded proteins interact and transactivate Jun family-dependent transcription via their atypical bZIP motif.

Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the role of HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins from other HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteins are limited to our previously reported study (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.

Prevalence and genetic characterisation of HTLV-1 and 2 dual infections in patients with pulmonary tuberculosis in Central-West Brazil.

Human T-cell lymphotropic virus (HTLV) may impact the clinical course of tuberculosis (TB). Both infections are highly endemic in Brazil. The aim of this study was to assess the prevalence of HTLV-1/2 in TB patients in Central-West Brazil and to perform a genetic characterisation of the respective isolates. Of the 402 patients, six (1.49%) were positive for anti-HTLV and five (1.24%; 95% confidence interval: 0.46-3.05) were infected with HTLV-1/2. Genetic characterisation demonstrated that the four HTLV-1 isolates belonged to the Transcontinental subgroup A of the Cosmopolitan subtype a and that the HTLV-2 isolate belonged to subtype a (HTLV-2a/c). The prevalence of HTLV infection observed in this study is higher than that observed in local blood donors and the HTLV-1 and 2 subtypes identified are consistent with those circulating in Brazil.

A quantitative polymerase chain reaction test to enumerate leukocytes in allograft tissue and the implications for donor eligibility testing.

A country-to-country analysis of infectious disease screening requirements for donated tissues or cells reveals they are not often harmonized. Transmission of one such infectious disease, human T-lymphotropic virus (HTLV), is related to the transfer of HTLV-infected, viable leukocytes of sufficient number. The ability to characterize allograft tissue as being absent of leukocytes, or containing relatively few leukocytes, by using a specific test has not been previously investigated. A quantitative polymerase chain reaction (qPCR) test was developed to interrogate protein tyrosine phosphatase, receptor type C (PTPRC) gene expression in tissue samples and was able to determine the number of leukocytes present in a tissue. The impact of a qualified leukocyte tissue testing method should be significant and lead to changes in donor eligibility regulations in certain countries. Human leukapheresis samples were used as a control to establish the amount of PTPRC in leukocytes. That value was used as a comparator to determine the number of leukocyte equivalents in tissues of interest. The qPCR test measured tissue leukocyte equivalents and the results were consistent with the relative abundance of leukocytes predicted for each tissue. Using qPCR to calculate leukocyte equivalents based upon PTPRC gene expression can be successfully employed to estimate the number of leukocytes in a tissue or allograft. This method could be used as a screen to rule out tissues that do not meet the criteria of being leukocyte rich and, therefore, do not need direct HTLV testing.

Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens.

BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100-1,000 geq/ml of plasma of HIV-1 subtypes (A - G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors' clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.

Human T-cell lymphotropic virus type 3 (HTLV-3)- and HTLV-4-derived antisense transcripts encode proteins with similar Tax-inhibiting functions but distinct subcellular localization.

The human T-cell lymphotropic virus (HTLV) retrovirus family is composed of the well-known HTLV type 1 (HTLV-1) and HTLV-2 and the most recently discovered HTLV-3 and HTLV-4. Like other retroviruses, HTLV-1 and HTLV-2 gene expression has been thought to be orchestrated through a single transcript. However, recent reports have demonstrated the unique potential of both HTLV-1 and HTLV-2 to produce an antisense transcript. Furthermore, these unexpected and newly identified transcripts lead to the synthesis of viral proteins termed HBZ (HTLV-1 basic leucine zipper) and APH-2 (antisense protein of HTLV-2), respectively. As potential open reading frames are present on the antisense strand of HTLV-3 and HTLV-4, we tested whether in vitro antisense transcription occurred in these viruses and whether these transcripts had a coding potential. Using HTLV-3 and HTLV-4 proviral DNA constructs, antisense transcripts were detected by reverse transcriptase PCR. These transcripts are spliced and polyadenylated and initiate at multiple sites from the 3' long terminal repeat (LTR). The resulting proteins, termed APH-3 and APH-4, are devoid of a typical basic leucine zipper domain but contain basic amino acid-rich regions. Confocal microscopy and Western blotting experiments demonstrated a nucleus-restricted pattern for APH-4, while APH-3 was localized both in the cytoplasm and in the nucleus. Both proteins showed partial colocalization with nucleoli and HBZ-associated structures. Finally, both proteins inhibited Tax1- and Tax3-mediated HTLV-1 and HTLV-3 LTR activation. These results further demonstrate that retroviral antisense transcription is not exclusive to HTLV-1 and HTLV-2 and that APH-3 and APH-4 could impact HTLV-3 and HTLV-4 replication.

HTLV infection among foreign pregnant women living in Spain.

BACKGROUND: The overall seroprevalence of HTLV infection among pregnant women in Spain is below 0.02% and accordingly universal antenatal screening is not recommended. However, as the number of immigrants has significantly increased during the last decade, this population might warrant specific considerations. OBJECTIVE: To evaluate the seroprevalence of HTLV infection among immigrant pregnant women living in Spain. METHODS: From January 2009 to December 2010 a cross-sectional study was carried out in all foreign pregnant women attended at 14 Spanish clinics. All were tested for HTLV antibodies using a commercial enzyme-immunoassay, being reactive samples confirmed by Western blot or PCR. RESULTS: A total of 3337 foreign pregnant women were examined. Their origin was as follows: Latin America 1579 (47%), North Africa 507 (16%), East Europe 606 (18%), Sub-Saharan Africa 316 (9%), North America and West Europe 116 (3.5%) and Asia and Australia 163 (5%). A total of 7 samples were confirmed as HTLV positive, of which 6 were HTLV-1 and 1 HTLV-2. HTLV-1 infection was found in 5 women coming from Latin America and 1 from Morocco. The only woman with HTLV-2 came from Ghana. The overall HTLV seroprevalence was 0.2%, being 0.3% among Latin Americans and 0.2% among Africans. It was absent among women coming from other regions. CONCLUSIONS: The seroprevalence of HTLV infection among foreign pregnant women in Spain is 0.2%, being all cases found in immigrants from Latin America and Africa. Given the benefit of preventing vertical transmission, antenatal screening should be recommended in pregnant women coming from these regions.

Human T-lymphotropic virus tax activates human cytomegalovirus major-immediate early promoter and improves production of recombinant proteins in HEK293 cells.

The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter.