Saiful Islam

HIV-1 unspliced RNA serves two distinct functions during viral replication: it is packaged into particles as the viral genome, and it is translated to generate Gag/Gag-Pol polyproteins required for virus assembly. Recent studies have demonstrated that in cultured cells, HIV-1 uses multiple transcription start sites to generate several unspliced RNA species, including two major transcripts with three and one 5′ guanosine, referred to as 3G and 1G RNA, respectively. Although nearly identical, 1G RNA is selected over 3G RNA to be packaged as the virion genome, indicating that these RNA species are functionally distinct. Currently, our understanding of HIV-1 transcription start site usage and the functions of RNA species is based on studies using cultured cells. Here, we examined samples from people living with HIV to investigate HIV-1 transcription start site usage and its impact on RNA function. Using paired samples collected from the same participants on the same date, we examined the HIV-1 unspliced RNA species in infected cells (PBMC) and in viruses (plasma). Our findings demonstrate that in people living with HIV, the virus uses multiple transcription start sites to generate several unspliced transcripts, including 3G and 1G RNA. Furthermore, we observed an enrichment of 1G RNA in the paired plasma samples, indicating a preferential packaging of 1G RNA in vivo . Our study illustrates the complex regulation of HIV-1 unspliced RNA in people living with HIV and provides valuable insights into how HIV-1 unspliced RNAs serve their functions in vivo . IMPORTANCE: HIV-1 virions must contain unspliced RNA and its translation products to maintain infectivity. How HIV-1 unspliced RNA fulfills these two essential and yet distinct roles in viral replication has been a long-standing question in the field. In this report, we demonstrate that in people living with HIV, the virus uses multiple transcription start sites to generate several unspliced RNA species that are 99% identical in sequence but differ functionally. One of the RNA species, 1G RNA, is selected over other HIV-1 unspliced RNAs to be packaged into viral particles. These findings are consistent with previous cell-culture-based observations and provide insights into how HIV-1 regulates its unspliced RNA function in people living with HIV.

HIV-1 relies on host RNA polymeraseII (Pol II) to transcribe its genome and uses multiple transcription start sites (TSS), including three consecutive guanosines located near the U3-R junction, to generate transcripts containing three, two, and one guanosine at the 5′ end, referred to as 3G, 2G, and 1G RNA, respectively. The 1G RNA is preferentially selected for packaging, indicating that these 99.9% identical RNAs exhibit functional differences and highlighting the importance of TSS selection. Here, we demonstrate that TSS selection is regulated by sequences between the CATA/TATA box and the beginning of R. Furthermore, we have generated two HIV-1 mutants with distinct 2-nucleotide modifications that predominantly express 3G RNA or 1G RNA. Both mutants can generate infectious viruses and undergo multiple rounds of replication in T cells. However, both mutants exhibit replication defects compared to the wild-type virus. The 3G-RNA-expressing mutant displays an RNA genome–packaging defect and delayed replication kinetics, whereas the 1G-RNA-expressing mutant exhibits reduced Gag expression and a replication fitness defect. Additionally, reversion of the latter mutant is frequently observed, consistent with sequence correction by plus-strand DNA transfer during reverse transcription. These findings demonstrate that HIV-1 maximizes its replication fitness by usurping the TSS heterogeneity of host RNA Pol II to generate unspliced RNAs with different specialized roles in viral replication. The three consecutive guanosines at the junction of U3 and R may also maintain HIV-1 genome integrity during reverse transcription. These studies reveal the intricate regulation of HIV-1 RNA and complex replication strategy.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4⁺ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro . Here we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo . A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental results and mathematical modeling demonstrated that addition of LRA to ART showed latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay can be used to optimize therapeutic against HIV-1 latency and investigate mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

Combination anti-retroviral therapy (cART) has drastically improved the clinical outcome of HIV-1 infection. Nonetheless, despite effective cART, HIV-1 persists indefinitely in infected individuals. Clonal expansion of HIV-1-infected cells in peripheral blood has been reported recently. cART is effective in stopping the retroviral replication cycle, but not in inhibiting clonal expansion of the infected host cells. Thus, the proliferation of HIV-1-infected cells may play a role in viral persistence, but little is known about the kinetics of the generation, the tissue distribution or the underlying mechanism of clonal expansion in vivo. Here we analyzed the clonality of HIV-1-infected cells using high-throughput integration site analysis in a hematopoietic stem cell-transplanted humanized mouse model. Clonally expanded, HIV-1-infected cells were detectable at two weeks post infection, their abundance increased with time, and certain clones were present in multiple organs. Expansion of HIV-1-infected clones was significantly more frequent when the provirus was integrated near host genes in specific gene ontological classes, including cell activation and chromatin regulation. These results identify potential drivers of clonal expansion of HIV-1-infected cells in vivo.

Recurrent cholera causes significant morbidity and mortality among the growing population of Dhaka, the capital city of Bangladesh. Previous studies have demonstrated that household contacts of cholera patients are at >100 times higher risk of cholera during the week after the presentation of the index patient. Our prospective study investigated the mode of transmission of Vibrio cholerae, the cause of cholera, in the households of cholera patients in Dhaka city. Of total 420 rectal swab samples analyzed from 84 household contacts and 330 water samples collected from 33 households, V. cholerae was isolated from 20%(17/84) of household contacts, 18%(6/33) of stored drinking water, and 27%(9/33) of source water samples. Phenotypic and molecular analyses results confirmed the V. cholerae isolates to be toxigenic and belonging to serogroup O1 biotype El Tor (ET) possessing cholera toxin of classical biotype (altered ET). Phylogenetic analysis by pulsed-field gel electrophoresis (PFGE) showed the V. cholerae isolates to be clonally linked, as >95% similarity was confirmed by sub-clustering patterns in the PFGE (NotI)-based dendrogram. Mapping results showed cholera patients to be widely distributed across 25 police stations with the highest incidence in households near the major rivers and polluted water bodies. The data presented on the transmission of infectious V. cholerae within the household contacts of cholera patients through drinking water underscores the need for safe water to prevent spread of cholera and related deaths in Dhaka city.

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Background: Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission ofVibrio choleraeassociated with 2012 cholera outbreaks in Nepal.Methods: V. cholerae(n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE).Results: AllV. choleraestrains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed theV. choleraestrains to possess the B-7 allele ofctxsubunit B. DNA sequencing oftcpArevealed a point mutation at amino acid position 64 (N → S) while thectxABpromoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'.V. choleraepossessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498–511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence ofgyrAandparCof the NA^Rstrains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the NepaleseV. choleraeto be clonal, and related closely withV. choleraeassociated with cholera in Bangladesh and Haiti.Conclusions: In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistantV. choleraeEl Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely withV. choleraeassociated with cholera in Bangladesh and Haiti.