The HIV-1 Tat Protein Enhances Splicing at the Major Splice Donor Site

ABSTRACTTranscription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5′ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5′ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and thetrans-acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.IMPORTANCEThe HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.

Encapsidation Determinants Located Downstream of the Major Splice Donor in the Maedi-Visna Virus Leader Region

ABSTRACT We investigated the role of the 5′-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Δ3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Δ5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Δ5MSD mutant and a double mutant (DM) (Δ5MSD and Δ3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Δ5MSD was dispensable in FOS cells, and the DM had the same REE as the Δ3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors.