Positive and Negative Modulation of Human Immunodeficiency Virus Type 1 Rev Function by cis and trans Regulators of Viral RNA Splicing

Abstract

Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. To investigate the possibility that modulation of viral RNA metabolism may alter Rev function, we analyzed the impact of multiple SR proteins on both processes. While overexpression of several of the SR factors altered splicing of HIV-1 env mRNA, they had disparate effects on Rev function that varied with the cell line used. Subsequent examination of exon splicing enhancer (ESE) and/or silencer (ESS) deletions suggests that the effects of the SR proteins on Rev function are not mediated through interaction with these elements. However, analysis of the deletions did indicate that the ESE and/or ESS does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. In situ hybridization and reverse transcription-PCR indicated that the loss of Rev response upon deletion of the ESE was due to a failure of Rev to induce transport of the unspliced RNA into the cytoplasm. Together, the data indicate that cellular splicing factors and viral regulatory elements can have significant stimulatory and inhibitory effects on Rev function, raising the possibility that cells can be rendered permissive or nonpermissive for virus replication by modulation of splicing activities.Human immunodeficiency virus type 1 (HIV-1) provides a useful model system for the study of RNA metabolism, as successful viral replication involves interplay between the processes of splicing and RNA transport. The primary viral RNA transcript is processed through alternative splicing into Ͼ25 different mRNA products (33) to generate the multiple proteins required for efficient virus assembly. The resulting viral mRNAs can be placed into three classes: the 9-kb unspliced RNA encoding gag and gag/pol proteins; the 4-kb singly spliced RNAs encoding env, vif, vpr, and vpu; and the 2-kb completely spliced RNAs which code for the regulatory proteins Tat, Rev, and Nef (45). In addition to undergoing splicing, viral RNAs of the 9-and 4-kb class are also exported from the nucleus in a process mediated by the interaction with the viral protein Rev (18, 29). The need for both splicing and Rev-mediated transport of the various viral RNAs suggests that these processes compete with one another. This hypothesis is supported by the observation that Rev function is dependent upon continued synthesis of viral RNA (20). If Revmediated export operates in competition with other RNA metabolic processes, then enhancing viral RNA splicing would be expected to inhibit HIV-1 replication by reducing the amount of incompletely spliced viral RNAs for Rev-mediated export and subsequent translation into structural proteins.In addition to being regulated by the suboptimal signals present within the 3Ј splice sites (3Јss) (28, 40), splicing of HIV-1 RNAs is also regulated by several cis-acting e...

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

“…Moreover, the ESE and ESS within the terminal exon also regulate viral RNA transport. Deletion of the ESE results in loss of Revinduced transport of the unspliced RNA to the cytoplasm (Pongoski et al 2002). The inhibition of Rev-induced RNA transport is ESS dependent because deletion of both the ESE and the ESS restores transport of the RNA.…”

Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

“…Retroviruses use many general SREs to regulate splicing of their RNA (for review, see Stoltzfus and Madsen 2006) and also use RNA elements to regulate nuclear export of unspliced RNA (for reviews, see Pollard and Malim 1998;Cullen 2003). How HIV RNAs balance between the two pathways is not completely clear, but some common elements might serve as a functional bridge between the two pathways by participating in the regulation of both splicing and RNA export (e.g., an ESS recognized by hnRNP A1 [Asai et al 2003] and the Rev response elements [Pongoski et al 2002]). …”

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

“…The combination of these various sites gives rise to at least 35 different mRNAs (1). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5Ј-terminal sequence (3,4), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Several studies have shown that HIV-1 acceptor sites are suboptimal as follows.…”

“…Although variations of the cellular concentrations of some of the SR proteins, namely SC35 and 9G8, have been observed after cell infection by virus HIV-1 (37,38), little is known about the action of SR proteins on the competition between the eight splicing acceptor sites and four splicing donor sites of HIV-1 RNA. The present knowledge in this field can be summarized as follows: (i) deep studies, which were performed both in vitro and in vivo, demonstrated the action of proteins ASF/SF2 and SC35 at acceptor site A7 (17,19,20,39,40); (ii) an in vitro study performed with S100 extract showed that, among the SR proteins SC35, SRp40, SRp55, and SRp70, only protein SC35 is able to activate site A3 in S100 extract (41). For a complete study of the effects of individual SR proteins on the entire set of HIV-1 splicing sites in cellular conditions, we cotransfected HeLa cells with a construct that produces a truncated HIV-1 RNA containing all the splicing sites (13,24), and a plasmid that overexpresses one of the SC35, SRp40, ASF/SF2, and 9G8 SR proteins.…”