15 research outputs found
Labeling HIV-1 nucleic acids during infection and effect of MOV10 in HBV replication
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.Includes bibliographical reference
Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus
RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action
A simplified quantitative real-time PCR assay for monitoring SARS-CoV-2 growth in cell culture
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growt
Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis
Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells
We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation
Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication
Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA, and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines, providing strong evidence for the involvement of MOV10 in the HBV life cycle. We demonstrated that MOV10 does not interact with HBV-core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. We conclude that the restriction of HBV by MOV10 is mediated through effects at the level of viral RNA
Comparative Investigation of the Genomic Regions Involved in Antigenic Variation of the TprK Antigen among Treponemal Species,Subspecies, and Strains
Although the three Treponema pallidum subspecies (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum), Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence. tprK heterogeneity is generated by nonreciprocal gene conversion between the tprK expression site and donor sites. Although each of the above-mentioned species and subspecies has a functional tprK antigenic variation system, it is still unclear why the level of expression and the rate at which tprK diversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as the tprK expression sites, among eight T. pallidum subsp. pallidum isolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), three T. pallidum subsp. pertenue isolates (Gauthier, CDC2, and Samoa D), one T. pallidum subsp. endemicum isolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain of T. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that the tprK recombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression of tprK variants among these Treponema isolates
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Multiplex single-cell visualization of nucleic acids and protein during HIV infection
Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop 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 (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional “bursts” of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents
The translational landscape of SARS-CoV-2-infected cells reveals suppression of innate immune genes
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development