Labeling HIV-1 nucleic acids during infection and effect of MOV10 in HBV replication

Abstract

Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we have developed a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV spliced and unspliced RNA, cytoplasmic and nuclear DNA, and Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We have also reported transcriptional "bursts" of nascent RNA from integrated proviral DNA. The major barrier towards a cure is the ability of the virus to remain dormant in long-lived cells known as the latent HIV reservoirs; however, the mechanisms that regulate latency are poorly understood. HIV-1 integration is directed to regions of the genome by the chromatin binding protein lens epitheliumderived growth factor (LEDGF/p75, referred to here as LEDGF). In the absence of LEDGF, HIV-1 infection is markedly less efficient. We report for the first time that knocking out LEDGF results in upregulation of antisense HIV-1 RNA transcription. Antisense HIV-1 transcripts are abundant in a higher proportion of cells when the Integrase-LEDGF interaction is lost, whether by knocking out LEDGF or by pharmacological inhibition. We also demonstrate that intriguingly, antisense RNA is rapidly lost from infected cells. Using a dual reporter virus to investigate the association between HIV-1 antisense RNA and latency, we have determined that antisense RNA has low expression in “latently” infected cells. Hepatitis B virus infection is a major health problem, there are more than 350 million people living with chronic HBV. Immune system can clear infection during the acute phase in immune-competent patients. A better understanding of the immune response against HBV infection may lead to new insights a find a cure. RNA helicases contribute to the immune response and some also have antiviral effects. MOV10 is a helicase RNA protein that has detrimental effect in HIV infection and HCV replication under MOV10 depletion or overexpression. Considering that HBV replication rely on reverse transcription to form DNA from pre-genomic RNA, we study the effect of MOV10 in HBV replication. Our data suggest that while MOV10 overexpression leads to HBV reduction, suppression of MOV10 enhances HBV replication.