Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production - PubMed (original) (raw)

Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production

Sandrine Jacquenet et al. Retrovirology. 2005.

Abstract

In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.

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Figures

Figure 1

Figure 1

HIV-1 splicing pattern. Schematic representation of HIV-1 proviral DNA. Open boxes represent the open reading frames encoding the viral proteins. Black boxes represent exons generated by combination of donor sites (D1 to D4) and acceptor sites (A1 to A7). The viral translation initiator codons are indicated by AUG.

Figure 2

Figure 2

Regulation of HIV-1 alternative splicing by SR proteins. Analysis of 2 kb (A) and 4 kb (B) mRNAs was performed by RT-PCR using 10 μg of total cellular RNA extracted from 293T cells transfected by HIV-1 pNL4.3 only (lane 1) or together with one SR plasmid (lanes 2–4). Viral mRNAs were identified according to the nomenclature of Purcell and Martin [2].

Figure 3

Figure 3

Influence of SR proteins on HIV-1 protein synthesis. 293T cells (2 × 105 per well) were transfected with 1 μg of HIV-1 pNL4.3 in the presence of increasing amounts of plasmid encoding either ASF/SF2, SC35 or 9G8. DNA concentrations were maintained constant by supplementation with the pCLacZ control plasmid which also served to monitor transfection efficiency. Values reported here correspond to assays carried out with a HIV to SR DNA molar ratio of 1:05. Cells were recovered two days after DNA transfection. A: Levels of Gag production were assessed by CAp24 antigen ELISA and expressed as pg of CA per μg of total cellular proteins. Note that ASF had a clear negative impact on Gag accumulation in cells whereas 9G8 had an opposite effect. B: Equivalent amounts of CAp24 antigen as measured by ELISA were subjected to western blotting. The same membrane was alternatively probed with the respective antibodies as indicated on the right: anti-CAp24 for Gag, anti-Vpr for p15, anti-NEF for p27 and anti-TMgp41 for Env. The viral Gag, Vpr, NEF and Env proteins are indicated according to their molecular weights in kDaltons. Note that SR proteins did not change the Gag processing pattern (compare lanes 2–4 and 1). ASF caused an indirect increase of Vpr cellular accumulation (lane 2) in agreement with its positive effect on Vpr mRNA level (Figure 1). On the other hand SC35 and 9G8 had an opposite effect (lanes 3–4). All Env levels were low (lanes 2–4) except in the control (lane 1).

Figure 4

Figure 4

Confocal microscopy of cells co-expressing HIV-1 Gag, Env and SR-protein. Panel A: 293T cells expressing HIV-1 pNL4.3 were subjected to immuno-staining using anti-Map17 (green staining) and anti-Env gp120 (red staining) antibodies and staining was viewed by confocal microscopy as described in methods. Most if not all cells expressed Gag and Env but only partial colocalization was seen (merge picture). Right panel corresponds to the same cells viewed by phase contrast microscopy. Panel B: same as in A except that His tagged-ASF/SF2 SR protein was overexpressed by DNA transfection with about 75% transfection efficiency (see methods). ASF/SF2 protein is localized in the nucleus (blue staining) and its overexpression caused a drastic reduction of Env level while Gag remained well expressed in agreement with the western blot data (Figure 3) but with an heterogenous pattern (first panel). Panel C: same as in A except that His tagged-SC35 SR protein was overexpressed by DNA transfection with about 75% transfection efficiency (see methods). SC35 protein (nuclear blue staining) overexpression caused a reduction of Env level while Gag was still highly expressed in agreement with the western blot data (Figure 2). Note that in all cases examined here (anti-Map17; green staining in panel A to C) Gag was found to accumulate at the plasma membrane and in intracellular compartments corresponding to vesicles [42] (Muriaux et al., unpublished data).

Figure 5

Figure 5

Influence of SR protein on cellular levels of HIV-1 Gag and Env. 293T cells expressing HIV-1 and one SR protein (either ASF, SC35 or 9G8) were immuno-stained, examined and counted using Confocal Laser Scanning Microscopy (see figure 4). Numbers are representative of more than 100 SR positive cells. For all experiments we evaluated the expression of Gag and Env, and SR protein when applicable. The numbers are expressed as the percentage of all SR positive cells given a DNA transfection efficiency of 70–75% (not shown). When HIV-1 pNL4.3 was transfected alone, 100% of the cells were found to co-express Gag and Env (first bar). Upon co-transfection with the ASF coding DNA, a majority of the cells only expressed ASF and about half of them expressed Gag and the SR protein (see ASF bars). Upon co-transfection of pNL4.3 and either the SC35 or 9G8 coding plasmid, a majority of cells expressed Gag and the SR protein (see SC35 and 9G8 bars).

Figure 6

Figure 6

Expression of viral proteins results from alterations of splicing pattern. 293T cells (2 × 105 per well) were transfected with 1 μg of HIV-1 pNL4.3 in the presence of increasing amounts of plasmid encoding either ASF/SF2, SC35 or 9G8 (ratios indicate molar amounts of HIV-1 DNA vs SR-expressing vector). DNA concentrations were maintained constant by supplementation with the pCLacZ control plasmid which also served to monitor transfection efficiency. A: Viral production was monitored by CAp24 antigen ELISA and expressed as ng of p24 per ml of medium (see methods). Results are representative of 3 independent experiments. Note that the effect of ASF/SF2 on virion production was already drastic at a HIV/SR molar ratio of 1:0.5. B: The pelleted viral particles were tested for their content in Gag, Pol, Env and Vpr proteins. Equivalent amounts of CAp24 antigen measured by ELISA were subjected to Western blotting. The same membrane was alternatively probed with the respective antibodies as indicated on the right: anti-CAp24 for Gag, anti-RT for p66 and p51, anti-Vpr for p15 and anti-TMgp41 for Env. The viral Gag, RT, Vpr and Env proteins are indicated according to their molecular weights in kDaltons. Note that fully mature CAp24 and RTp66/p51 were abundant in all virion preparations. ASF caused an indirect increase of Vpr incorporation in virions (lane 2) whereas SC35 and 9G8 had an opposite effect (lanes 3–4). All Env levels were low (lanes 2–4) except in the control (lane 1).

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References

    1. Frankel AD, Young JA. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem. 1998;67:1–25. doi: 10.1146/annurev.biochem.67.1.1. - DOI - PubMed
    1. Purcell DF, Martin MA. Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol. 1993;67:6365–6378. - PMC - PubMed
    1. Felber BK, Hadzopoulou-Cladaras M, Cladaras C, Copeland T, Pavlakis GN. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989;86:1495–1499. - PMC - PubMed
    1. Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989;338:254–257. doi: 10.1038/338254a0. - DOI - PubMed
    1. Damier L, Domenjoud L, Branlant C. The D1-A2 and D2-A2 pairs of splice sites from human immunodeficiency virus type 1 are highly efficient in vitro, in spite of an unusual branch site. Biochem Biophys Res Commun. 1997;237:182–187. doi: 10.1006/bbrc.1997.7091. - DOI - PubMed

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