Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

Abstract

Alternative splicing of the primary transcript plays a key role in retroviral gene expression. In contrast to all known mechanisms that mediate alternative splicing in retroviruses, we found that in murine leukemia virus, distinct elements located upstream of the 5 splice site either inhibited or activated splicing of the genomic RNA. Detailed analysis of the first untranslated exon showed that the primer binding site (PBS) activates splicing, whereas flanking sequences either downstream or upstream of the PBS are inhibitory. This new function of the PBS was independent of its orientation and primer binding but associated with a particular destabilizing role in a proposed secondary structure. On the contrary, all sequences surrounding the PBS that are involved in stem formation of the first exon were found to suppress splicing. Targeted mutations that destabilized the central stem and compensatory mutations of the counter strand clearly validated the concept that murine leukemia virus attenuates its 5 splice site by forming an inhibitory stem-loop in its first exon. Importantly, this mode of splice regulation was conserved in a complete proviral clone. Some of the mutants that increase splicing revealed an opposite effect on translation, implying that the first exon also regulates this process. Together, these findings suggest that sequences upstream of the 5 splice site play an important role in splice regulation of simple retroviruses, directly or indirectly attenuating the efficiency of splicing.A characteristic feature of all retroviruses is the process of reverse transcription of the RNA genome into double-stranded DNA. Following integration into the host genome, the proviral DNA functions as one expression unit, which is transcribed by cellular RNA polymerase II, yielding a single polycistronic primary transcript that serves as genomic RNA for progeny virus. Productive infection and formation of new retroviral particles require the well balanced expression of all viral genes. This is accomplished by a combination of alternative splicing (intron retention) and regulated nuclear export of the primary transcript on the RNA processing level and proteolytic cleavage and translational read-through on the post-translational level (reviewed in Refs. 1-4).The genomic organization of all retroviruses is similar (Fig. 1A). The gag-pol open reading frame (ORF) 3 encoding the inner structural proteins (Gag), and the replication enzymes (Pol) is located in the 5Ј half of the transcript and expressed from the unspliced genomic RNA after nuclear export. The gag-pol ORF in all primary retroviral transcripts is defined as an intron through the presence of a preceding 5Ј splice site (ss) in the 5Ј-untranslated region and a functional 3Јss located toward the end of the polymerase ORF (Fig. 1A). To express the glycoproteins (Env), which are encoded in the 3Ј half of the genomic RNA, the gag-pol ORF is removed by a single splice event for subsequent export of the fully spliced RNA (1, 3, 4). This onesplice event strategy cre...

Murine Leukemia Virus Uses NXF1 for Nuclear Export of Spliced and Unspliced Viral Transcripts

“…Previous studies have demonstrated that modulation of the viral 5= leader sequence affects the cytoplasmic accumulation of spliced and unspliced MLV transcripts. For instance, deletion of the first 28 nt of the R region is critical for the cytoplasmic level of unspliced full-length viral transcripts (20,64), while deletions in the PBS sequence also modulate the balance between unspliced and spliced MLV transcripts (21).…”

“…Previous studies have demonstrated the importance of the 5= UTR for the alternative splicing of MLV transcripts; the PBS activates splicing, while flanking sequences downstream of the PBS (3= PBS) are inhibitory (21). We generated the MLV splicing mutants pAMS⌬PBS and pAMS⌬3=PBS based on the pAMS plasmid (Fig.…”

“…For expression of the viral glycoprotein Env, the Gag-and Pol-coding region is removed as an intron by a single splice event between the SD site and the splice acceptor (SA) site located upstream of the Env start codon. Previous studies have demonstrated that the sequences upstream of the 5= major SD site determine the splicing efficiency of MLV transcripts (20,21). However, the nuclear export pathway(s) and the mechanism controlling the cytoplasmic accumulation of viral transcripts remain elusive.…”

Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer

“…Gammaretroviral vectors termed SF91 were derived from SF91.GFP (11,31). The vector SF91aPBS.GFP.pre was generated by introducing the artificial primer binding site (aPBS) as an XbaI/ApaI fragment from SF91aPBS.GFP (8,15) in SF91.GFP.pre (31). The retroviral vector used for engineering human Cre reporter cells to express Fv1 n was derived from pCFCR (21).…”

Use of human MAR elements to improve retroviral vector production

“…17,18 This has been associated to an abundant transcription from the internal promoter driving transgene expression, which may occur at the expense of full-length viral RNA transcription originating from the LTR in packaging cells. 18,19 Various approaches were taken to overcome these limitations, such as the introduction of the post-transcriptional element from Woodchuck hepatitis virus into the 3¢-untranslated region of SIN vectors, 20,17 or the incorporation of upstream polyadenylation enhancer elements derived from viral or cellular genes into the 3¢U3 region of the LTR, allowing partial recovery of high viral vector titers. 21 Recently, it has been observed that the use of a strong internal enhancer/promoter in SIN vectors may increase transgene expression but that this may also increase host cell transformation.…”