A newly identified interaction between nucleolar NPM1/B23 and the HTLV-I basic leucine zipper factor in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.

Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries.

Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15-145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec, Canada, in 2017.

Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks. However, the extraction of norovirus RNA from frozen raspberries remains challenging. Recovery yields are low and PCR inhibitors limit the sensitivity of the detection methodologies. In 2017, 724 people from various regions of the Province of Quebec, Canada, were infected by noroviruses and the outbreak investigation pointed to frozen raspberries as a putative source. A new magnetic silica bead approach was used for the extraction of viruses from different outbreak samples. The RNA extracts were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and five samples were confirmed positive for norovirus by RT-qPCR amplicon sequencing. A multiplex long-range two-step RT-PCR approach was developed to amplify norovirus ORF2 and ORF3 capsid genes from the positive frozen raspberry RNA extracts and other sequencing strategies were also explored. These capsid genes were sequenced by Next-Generation Sequencing. Phylogenetic analyses confirmed the presence of multiple genotypes (GI.3, GI.6, and GII.17) and intra-genotype variants in some of the frozen raspberry samples. Variants of genotype GI.3 and GI.6 had 100% homology with sequences from patient samples. Similar strains were also reported in previous outbreaks. Confirmation approaches based on sequencing the norovirus capsid genes using Next-Generation Sequencing can be applied at trace level contaminations and could be useful to assess risk and assist in source tracking.

[Antisense proteins of HTLV viruses]. / Les protéines antisens des virus HTLV.

There are four human T-lymphotropic viruses (HTLV-1, 2, 3, 4) that have emerged from the transmission of simian viruses. HTLV-1 was the first retrovirus to be shown to be responsible for a human pathology. The expression of retroviral genes depends mostly on their 5'LTR, but it was revealed that HTLV have a promoter in their 3'LTR, capable of transcription from the antisense strand of their genome. These transcripts can be translated into proteins named HBZ, APH-2, APH-3 and APH-4. Antisense transcription in HTLV-1 and its encoded protein HBZ have been thoroughly studied and it has been suggested that HBZ plays an important role in viral replication and the development of ATL. Very few studies have been conducted on antisense transcription from the three other viruses, although it is likely that these genes are also implicated in viral replication.

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.

Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.

A new luciferase-based quantitative assay for the evaluation of human trophoblast fusion.

The syncytiotrophoblast is a multinuclear cell layer maintained through fusion events with cytotrophoblasts and plays a key role in the properties of the placenta. Monitoring fusion in this cell layer is important in studies aimed at understanding its function. We herein propose a new fusion assay based on the transactivating potential of the human immunodeficiency virus type 1 (HIV-1) Tat protein on its promoter present in the long terminal repeat (LTR) region. We used 2 BeWo cell populations, one stably transfected with the HIV-1 LTR positioned upstream of the luciferase gene and the other stably transfected with a Tat expression vector. Both stable cell lines were responsive to Tat-mediated LTR transactivation and demonstrated normal fusion and human chorionic gonadotropin (hCG) secretion upon stimulation. When both BeWo cell lines were cocultured, forskolin-mediated induction of fusion led to an increase in luciferase activity, which was sensitive to anti-syncytin 1 and -2 antibodies and syncytin 2 small interfering RNAs (siRNAs). Similar results were obtained in primary trophoblasts. Our results highlight the effectiveness and accuracy of this new quantification assay for trophoblast fusion.

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.