Biol. 19:69C77 [PMC free article] [PubMed] [Google Scholar] 40. be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of mRNA expression. We DNA2 inhibitor C5 further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. INTRODUCTION Replication of human immunodeficiency virus type 1 (HIV-1) is counteracted by four major classes of host-encoded restriction factors: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), TRIM5 (tripartite motif 5), tetherin (BST-2, CD317, or HM1.24), and SAMHD1 (1C4). A3G (5) belongs to a family of cytidine deaminases that includes seven members (A3A to A3D and A3F to A3H) located in a gene cluster on chromosome 22 (6C8). It is encapsidated into newly assembled virions and introduces C-to-U substitutions during minus-strand synthesis, resulting in G-to-A hypermutations and aberrant DNA ends DNA2 inhibitor C5 in the HIV-1 genome. Furthermore, A3G, independent of its deaminase activity, inhibits reverse transcriptase-mediated minus-strand elongation by direct binding to the viral RNA (9). This leads to massive impairment of viral replication (10). However, the HIV-1-encoded accessory protein Vif counteracts A3G by direct interaction, by inducing proteasomal degradation, and by repression of mRNA synthesis (10). Whereas HIV-1 is able to replicate efficiently in A3G-expressing cells, Vif-deficient virus strains are completely suppressed (5). Nevertheless, a narrowly restricted level of Vif is crucial for optimal HIV-1 replication since proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site is inhibited DNA2 inhibitor C5 by high levels of Vif (11). During the course of infection, the HIV-1 9-kb single-sense pre-mRNA is processed into more than 40 alternatively spliced mRNA isoforms encoding 18 HIV-1 proteins, all of which interact with a wide variety of host cell proteins, complexes, and pathways (12). Furthermore, negative-sense mRNAs that lead to the expression of at least one antisense protein are also found in HIV-1-infected T cells (13). The splicing process consists of DNA2 inhibitor C5 a series of consecutive steps that are orchestrated by interactions of individual spliceosomal components (14). The initial binding of the U1 snRNP to the pre-mRNA is mediated by base pairing between the 5 end of the U1 snRNA and the 5 splice site (5ss) initiating early E-complex formation (15). Subsequently, the 3ss, consisting of the AG dinucleotide, the branch point sequence (BPS), and a polypyrimidine tract, is bound by the U2 snRNP at the BPS in an ATP-dependent manner, thus initiating A-complex formation. In a process named exon definition, U1 and U2 snRNPs bound to the exon-intron borders pair with each other (cross-exon interactions) and facilitate the removal of the flanking introns (16, 17). Rev expression initiates the switch from the early to the late stage of viral replication (18). On binding to DNA2 inhibitor C5 the Rev-responsive element (RRE), Rev oligomerizes cooperatively and, by interacting with the cellular Crm1 export pathway, facilitates the export of intron-containing viral mRNAs (19C21). Since the AUG is localized within intron 2, this intron must be retained within the functional mRNA. Thus, the 5ss D2 has to be rendered splicing incompetent, even though binding of U1 snRNP to this 5ss is a prerequisite for efficient recognition of the upstream 3ss A1 (22, 23). In general, 5ss splice donor repression may be a common requirement for RYBP the generation of all spliced but intron-containing HIV-1 mRNAs, e.g., the mRNAs (24). Indeed, recent studies demonstrated that the splicing regulatory element (SRE)-mediated binding of the U1 snRNP to a 5ss does not necessarily result in its processing into the spliceosomal A-complex formation but can lead to a dead-end complex formation that prevents the splicing process (25C28). Comparative analyses of the intrinsic strengths of all HIV-1 3ss have shown that A1 is intrinsically inefficient (18) and, in the absence of other effects, would result in mRNA species representing only 1% of the total 4.0-kb mRNA class (29). However, in the presence of its downstream exonic sequence, A1.