PSF Acts through the Human Immunodeficiency Virus Type 1 mRNA Instability Elements To Regulate Virus Expression

Abstract

Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms. This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs. Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS. Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm, suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.Many eukaryotic mRNAs are subject to regulated turnover via cis-acting signals which are recognized by trans-acting factors. The best-studied example is the cytoplasmic mRNA decay mediated by cis-acting AU-rich RNA elements and trans-acting ARE-binding proteins. Another important mechanism is nonsense-mediated mRNA decay, which serves to eliminate the transcripts that contain premature stop codons (for recent reviews, see references 17, 34, and 40).In contrast to cytoplasmic pathways, nuclear mRNA turnover is less well understood. Among the mRNAs that are subject to nuclear downregulation are the Rev-responsive element (RRE)-containing mRNAs of human immunodeficiency virus type 1 (HIV-1) (11,22,25,26,32,33,42,48,54; reviewed in references 13, 21, 27, and 47). These unspliced and nonterminally spliced transcripts need to be exported from the nucleus before completion of splicing to produce the Gag-Pol and Env proteins. This regulatory step is accomplished by the viral Rev protein, which binds to the RRE and links these transcripts to the CRM1 export receptor. In the absence of Rev, these mRNAs are further spliced to completion or degraded. However, even when devoid of splice sites, the unspliced or nonterminally spliced mRNAs are poorly expressed due to the presence of cis-acting instability elements (INS/ CRS) that are scattered throughout the gag/pol and env mRNAs (11,25,32,42,50,51,54). These elements act at several steps, impairing mRNA stability, nucleocytoplasmic transport, and translation (5,15,22,29,33,51,54), whereas Rev counteracts these defects, resulting in efficient expression. However, Rev is unable to export nonterminally spliced mRNAs that do not contain a functional INS (51), and hence, INS are an integral part of Rev regula...

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

“…40 Lastly, PSF has been shown to repress the expression of HIV-1 gag/pol and env mRNAs through cis-acting instability elements (INS) that target degradation. 41 Previously, we showed that PSF and p54 nrb interact with U5 snRNA. 23 During further analysis of this interaction by immunoprecipitation (IP) with PSF-specific antibodies, we show here that all five U snRNPs co-IP with PSF when HeLa nuclear extracts are adjusted to splicing conditions, regardless of the addition of pre-mRNA.…”

Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4+T Cells

“…A previous study reported that PSF and NonO interact with the HIV-1 gag cis-acting instability elements (INS) and that overexpression of PSF, but not NonO, inhibits HIV-1 Gag p24 production in transfected HEK293 cells. 25 This study suggests that PSF and NonO may regulate HIV-1 mRNA production causing diminished HIV-1 gag-pol and env gene expression. 25 NonO has also been shown to form a complex with PSF and participate in the nonhomologous end-joining pathway, capable of DNA double-strand break rejoining.…”

“…25 This study suggests that PSF and NonO may regulate HIV-1 mRNA production causing diminished HIV-1 gag-pol and env gene expression. 25 NonO has also been shown to form a complex with PSF and participate in the nonhomologous end-joining pathway, capable of DNA double-strand break rejoining. 26 We thus speculated that NonO might have a role in the assembly of PICs and integration of the HIV-1 genome into the host chromosome.…”

“…The only study investigating NonO in HIV-1 gene expression found that the INS of HIV-1 gag assembled with PSF and NonO in nuclear extracts of cells, hinting at a possible role at later stages of HIV-1 infection. 25 However, only overexpression of PSF inhibited INS mRNA expression and extracellular Gag production in HEK293 cells. ) were infected at an equivalent MOI of 0.5 for 2 h. Cells were then washed and replated in fresh media and cultured for a total of 3 days.…”

Structural Study on Apoptosis of Chronic Eosinophilic Leukemia Cells by Interaction of S100A8 with Splicing Factor, Proline and Glutamine-Rich

“…Furthermore, it binds PTB1, non-POU-domain-containing octamer-binding protein (NONO), and cell division cycle 5-like (CDC5L). In addition, SFPQ is involved in the pathogenic pathway of influenza virus and human immunodeficiency virus (LanderasBueno et al, 2011;Zolotukhin et al, 2003). Especially, SFPQ forms splicing factor-rich foci in a perinuclear pattern, related to the differentiation of myeloid cells (Shav-Tal et al, 2001).…”