Abstract
ABSTRACTNullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.
Highlights
Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes
ZSG1 and Jurkat-ZSG1 cells were obtained by transduction of Jurkat cells with NB-ZSG1 and ZSG1 virus-like particles (VLPs) and isolation by fluorescence-activated cell sorting (FACS)
The results showed the occupancy of RNA polymerase II (RNAPII) on nuc-1 in NB-ZSG1treated, HIV-1-infected Jurkat cells to be very low and near the level of sensitivity of the chromatin immunoprecipitation (ChIP) assay, whereas a 48-fold enrichment of RNAPII occupancy on nuc-1 was measured in samples prepared from ZSG1-treated, HIV-1-infected Jurkat cells (Fig. 5B)
Summary
Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells. IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces measurable viral loads in patients below detectable levels. We show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, can inhibit HIV-1 transcription in infected Jurkat T cell to undetectable levels. Infected cells in patients may not express HIV-1 at all or express it at very low levels, but virus production can be increased by activation of the HIV-1 long terminal repeat (LTR) promoter by mechanisms not completely defined. Can be suppressed in a manner that establishes latent but replication-competent provirus in resting memory CD4ϩ T cells by mechanisms not yet fully defined
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