Long-term inhibition of HIV-1 replication with RNA interference against cellular cofactors. Antiviral Res
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Antiviral research
(Impact Factor: 3.94).
01/2011; 89(1):43-53. DOI: 10.1016/j.antiviral.2010.11.005
In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.
Available from: PubMed Central
- "As expected, our results for the HIV-1 control (Figure 1A) agree well with previous reports [57,59,62,77]. Depletion of ALIX modestly reduced virion release and infectivity (compare lane 3 to lanes 1 and 2, 3-fold reduction in virion release and 2-fold reduction in infectivity), whereas greater reductions were seen upon depletion of TSG101 (lane 4, 21-fold reduction in virion release and 101-fold reduction in infectivity), CHMP2A/B (lane 5, 18- and 101-fold reductions, respectively), CHMP4A/B (lane 6, 3- and 55-fold reductions, respectively) and VPS4A/B (lane 7, 2- and 12-fold reductions, respectively). "
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ABSTRACT: Retroviruses and many other enveloped viruses usurp the cellular ESCRT pathway to bud from cells. However, the stepwise process of ESCRT-mediated virus budding can be challenging to analyze in retroviruses like HIV-1 that recruit multiple different ESCRT factors to initiate budding.
In this study, we characterized the ESCRT factor requirements for budding of Equine Infectious Anemia Virus (EIAV), whose only known direct ESCRT protein interaction is with ALIX. siRNA depletion of endogenous ESCRT proteins and "rescue" experiments with exogenous siRNA-resistant wild type and mutant constructs revealed budding requirements for the following ESCRT proteins: ALIX, CHMP4B, CHMP2A and VPS4A or VPS4B. EIAV budding was inhibited by point mutations that abrogate the direct interactions between ALIX:CHMP4B, CHMP4B:CHMP2A, and CHMP2A:VPS4A/B, indicating that each of these interactions is required for EIAV budding. Unexpectedly, CHMP4B depletion led to formation of multi-lobed and long tubular EIAV virions.
We conclude that EIAV budding requires an ESCRT protein network that comprises EIAV Gag-ALIX-CHMP4B-CHMP2A-VPS4 interactions. Our experiments also suggest that CHMP4B recruitment/polymerization helps control Gag polymerization and/or processing to ensure that ESCRT factor assembly and membrane fission occur at the proper stage of virion assembly. These studies help establish EIAV as a streamlined model system for dissecting the stepwise processes of lentivirus assembly and ESCRT-mediated budding.
Retrovirology 10/2013; 10(1):104. DOI:10.1186/1742-4690-10-104 · 4.19 Impact Factor
Available from: ncbi.nlm.nih.gov
- "However, most putative HDFs identified by such approaches have yet to be validated in cells that are naturally infected by HIV. This is necessary as reporter cell lines may be misleading with respect to HDF importance, as exemplified in a study where only half of putative HDFs were validated as such in a T cell-derived line
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Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target.
shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels.
This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.
Virology Journal 11/2012; 9(1):272. DOI:10.1186/1743-422X-9-272 · 2.18 Impact Factor
Available from: Sophie Borel
- "Besides its role as an intracellular host defense mechanism against viruses, autophagy can also be used by the virus for its own profit to replicate more efficiently in cells, or to control cell survival (Espert et al., 2007; Deretic and Levine, 2009; Espert and Biard-Piechaczyk, 2009; Lunemann and Munz, 2009; Lin et al., 2010; Sumpter and Levine, 2010). Data from a genome-wide RNAi screen (Brass et al., 2008) and silencing of 30 candidate cofactors (Eekels et al., 2011) indicate that HIV-1 replication in cells requires the presence of several Atg (Atg7, GABARAPL2, Atg12, and Atg16L). A very recent study underlines the role of several Atgs, in particular Atg5 and Atg16, in HIV-1 replication in CD4+ T cells (Eekels et al., 2012). "
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ABSTRACT: Autophagy is an intracellular mechanism whereby pathogens, particularly viruses, are destroyed in autolysosomes after their entry into targets cells. Therefore, to survive and replicate in host cells, viruses have developed multiple strategies to either counteract or exploit this process. The aim of this review is to outline the known relationships between HIV-1 and autophagy in CD4+ T lymphocytes and macrophages, two main HIV-1 cell targets. The differential regulation of autophagy in these two cell-types is highlighted and its potential consequences in terms of viral replication and physiopathology discussed.
Frontiers in Immunology 05/2012; 3:97. DOI:10.3389/fimmu.2012.00097
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