The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Y) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Y+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Y-RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Y does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Y+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Y thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag. Human immunodeficiency virus-1 (HIV-1), the major cause of the AIDS epidemic, is a retrovirus of the lentivirus family. Integrated into the infected cell genome as a provirus, it is transcribed by RNA polymerase II. Through a complex early and late genetic switch, controlled by the accessory protein Rev (1), it produces large numbers of budding virus particles each containing the diploid RNA genome. The identification and capture of viral RNA is remarkably specific. It is achieved by the uncleaved Gag polyprotein of the virus (2), binding to the unspliced genomic RNA species through initial high-affinity interactions with a complex stem loop structure in the viral leader region (3). The two dimensional (4,5) and three dimensional (6-8) structures of this packaging signal region (C) have been extensively studied and, although in HIV-1 a single discrete encapsidation motif, both necessary and sufficient for packaging has not been identified (9), the consensus is that the major signal is in a stem loop region called SL3 upstream of the Gag start codon (5). This is in contrast to the situation in murine (simple) retroviruses in which a distinct RNA region has been identified as necessary and sufficient (10), and it suggests that the RNA protein-recognition process in complex retroviruses is governed by additional factors. Mutations and/or deletions affecting SL3 have been shown to produce a packaging defect (3,11,12), in which near normal numbers of virions are produced from transfected or infected cells. These however have a profound replication defect and are found to contain severely reduced quantities of genomic RNA per virion. Specific Gag binding, initiating on SL3, is likely followed by recruitment o...
Summary There is an urgent need for rapid SARS-CoV-2 testing in hospitals to limit nosocomial spread. We report an evaluation of point of care (POC) nucleic acid amplification testing (NAAT) in 149 participants with parallel combined nasal and throat swabbing for POC versus standard lab RT-PCR testing. Median time to result is 2.6 (IQR 2.3–4.8) versus 26.4 h (IQR 21.4–31.4, p < 0.001), with 32 (21.5%) positive and 117 (78.5%) negative. Cohen’s κ correlation between tests is 0.96 (95% CI 0.91–1.00). When comparing nearly 1,000 tests pre- and post-implementation, the median time to definitive bed placement from admission is 23.4 (8.6-41.9) versus 17.1 h (9.0–28.8), p = 0.02. Mean length of stay on COVID-19 “holding” wards is 58.5 versus 29.9 h (p < 0.001). POC testing increases isolation room availability, avoids bed closures, allows discharge to care homes, and expedites access to hospital procedures. POC testing could mitigate the impact of COVID-19 on hospital systems.
Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4 + T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4 + T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.
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