Abstract
Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.
Highlights
§ Fellow of the French Ministere de la Jeunesse, de l’Education Nationale et de la Recherche
Several studies have shown that HIV-1 acceptor sites are suboptimal as follows. (i) Their polypyrimidine tracts are short and interrupted by purines [3, 5, 9]. (ii) Their branch point sequences are not canonical, and in some cases, a residue other than an adenosine is used as the branch site [22, 23]. (iii) Their accessibility is limited by their sequestering in stable secondary structures [24]. (iv) Several cis-inhibitory elements have been identified that down-regulate the A2, A3, and A7 sites by binding of hnRNP A1 or hnRNP H proteins [11, 12, 14, 16, 18, 19, 21]
Strategy Used for in Vitro Splicing Assays—As regulation of alternative splicing of HIV-1 RNA was proposed to depend mainly on the suboptimal properties of acceptor sites, the in vitro studies were focused on the effect of SR proteins on acceptor sites
Summary
§ Fellow of the French Ministere de la Jeunesse, de l’Education Nationale et de la Recherche. On the basis of the in vitro data (Fig. 1), the important increase found for vpr1 mRNA and the slight effects on nef4, nef5, and tat4 mRNAs can result from the strong activation of site A2 by protein ASF/SF2.
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