Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

RNA splicing at human immunodeficiency virus type 1 3' splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element.

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H.

An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3'-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 approximately 60 nucleotides downstream of 3'-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5' extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3'-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two U-to-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3'-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF(35) for binding to the exon sequence flanking 3'-splice site A3. This binding results in the inhibition of splicing at 3'-splice site A3.

Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site.

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) that, when used in conjunction with the regular HIV 3' ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.

Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

Selective inhibition of splicing at the avian sarcoma virus src 3' splice site by direct-repeat posttranscriptional cis elements.

The direct-repeat elements (dr1) of avian sarcoma virus (ASV) and leukosis virus have the properties of constitutive transport elements (CTEs), which facilitate cytoplasmic accumulation of unspliced RNA. It is thought that these elements represent binding sites for cellular factors. Previous studies have indicated that in the context of the avian sarcoma virus genome, precise deletion of both ASV dr1 elements results in a very low level of virus replication. This is characterized by a decreased cytoplasmic accumulation of unspliced RNA and a selective increase in spliced src mRNA. Deletion of either the upstream or downstream dr1 results in a delayed-replication phenotype. To determine if the same regions of the dr1 mediate inhibition of src splicing and unspliced RNA transport, point mutations in the upstream and downstream elements were studied. In the context of viral genomes with single dr1 elements, the effects of the mutations on virus replication and increases in src splicing closely paralleled the effects of the mutations on CTE activity. For mutants strongly affecting CTE activity and splicing, unspliced RNA but not spliced RNA turned over in the nucleus more rapidly than wild-type RNA. In the context of wild-type virus containing two dr1 elements, mutations of either element that strongly affect CTE activity caused a marked delay of virus replication and a selective increase in src splicing. However, the turnover of the mutant unspliced RNA as well as the spliced mRNA species did not differ significantly from that of the wild type. These results suggest the dr1 elements in ASV act to selectively inhibit src splicing and that both elements contribute to the fitness of the wild-type virus. However, a single dr1 element is sufficient to stabilize unspliced RNA.

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains.

In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.

Overlapping cis sites used for splicing of HIV-1 env/nef and rev mRNAs.

Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5' and 3' splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3' splice site (A5) is immediately downstream of the rev initiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3' branch points overlap with the AG dinucleotides at rev 3' splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3' splice site A4b AG dinucleotide dramatically increased splicing of the env/nef 3' splice site A5. This correlated with increased usage of the four most 3' branch points, which include those within the rev 3' splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nef branch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5' rev 3' splice site A4c with preferential use of the two most 5' branch points. Our results suggest that spliceosomes formed at rev A4a-4b, rev A4c, and env/nef A5 3' splice sites each recognize different subsets of the eight branch point sequences.

The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly.

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.

The upstream, direct repeat sequence of Prague A Rous sarcoma virus is deficient in mediating efficient Gag assembly and particle release.

Rous sarcoma virus (RSV) contains two approximately 135-nt imperfect direct repeats composed of smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt), that are between the env and src genes and downstream of src in the 3' untranslated region, respectively. It has previously been shown that a Prague A RSV mutant in which both dr1 sequences are deleted is defective at several points in the virus life cycle, including unspliced RNA and env mRNA stability, unspliced RNA transport, and virus particle assembly. A defect in unspliced RNA transport occurs because a cytoplasmic transport element is present within the dr1. We have suggested that the defect of particle production may arise from the failure of the unspliced RNA to be targeted to sites in the cytoplasm where its translation is favorable for Gag protein assembly. In this report, we have further investigated the function of the direct repeats by comparing virus mutants containing either a single upstream or downstream dr1 sequence. Both mutants were delayed in replication compared to the wild-type; the mutant with a single upstream dr1 (delta DDR) is significantly more defective than the mutant with a single downstream dr1 (delta UDR). While both mutants appear capable of efficiently transporting unspliced RNA to the cytoplasm, the delta DDR mutant with only the upstream dr1 is defective in its ability to support Gag assembly and particle release. The replication defect cannot be repaired by placing the upstream dr1 at the location of the downstream dr1 in the 3' untranslated region. A single point mutation in the upstream dr1 (U to C) restored replication and particle production to near normal levels. The results suggest that unspliced RNA transport and Gag assembly functions may be mediated by different elements within the dr1 and that the Prague A upstream dr1 is defective in the latter but not the former function.

Rous sarcoma virus direct repeat cis elements exert effects at several points in the virus life cycle.

Two approximately 135-nucleotide (nt) direct repeats flank the Rous sarcoma virus (RSV) oncogene src and are composed of two smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt). These sequences have been reported to contain cis-acting signals necessary for RNA packaging and elements that allow cytoplasmic accumulation of unspliced RNA (cytoplasmic transport elements). In this report, we show that avian fibroblasts infected with the Prague A strain of RSV with precise deletions of both dr1 elements express src and are transformed by this mutant virus but production of virus particles is very low and virus spread throughout the culture requires several weeks. We show that the replication defect is due to complex effects on viral RNA transport, viral RNA half-life, and virus particle assembly. The dr1 elements may contain binding sites for a permissive cell-specific factor(s) that facilitates efficient nuclear-cytoplasmic transport, RNA stability, and cytoplasmic utilization of unspliced viral RNA. The implications of these results for understanding the defects of nonpermissive virus infections in mammalian cells are discussed.

Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2.

We have previously demonstrated that an exon splicing silencer (ESS) is present within human immunodeficiency virus type 1 (HIV-1)tat exon 2. This 20 nucleotide (nt) RNA element acts selectively to inhibit splicing at the upstream 3'splice site (3'ss #3) flanking this exon. In this report, we have used in vitro splicing of mutated RNA substrates to determine the sequences necessary and sufficient for the activity of the ESS. The activity of the ESS within tat exon 2 maps to a 10 nt core sequence CUAGACUAGA. This core sequence was sufficient to inhibit splicing when inserted downstream from the 3'ss of the heterologous Rous sarcoma virus src gene. Mutagenesis of the interspersed purines in the polypyrimidine tract of the tat exon 2 3'ss to pyrimidines resulted in a significant increase in splicing efficiency indicating that 3'ss#3 is suboptimal. The ESS acts to inhibit splicing at the optimized 3'splice sites of both the HIV-1 tat and RSV src constructs but with a reduced efficiency compared to its effect on suboptimal 3'splice sites. The results indicate that both the ESS and a suboptimal 3'splice site act together to control splicing at the 3'splice site flanking at exon 2.

Selection and characterization of replication-competent revertants of a Rous sarcoma virus src gene oversplicing mutant.

All retroviruses require both unspliced and spliced RNA for a productive infection. One mechanism by which Rous sarcoma virus achieves incomplete splicing involves suboptimal env and src 3' splice sites. We have previously shown that mutagenesis of the nonconsensus src polypyrimidine tract to a 14-nucleotide uninterrupted polypyrimidine tract results in an oversplicing phenotype and a concomitant defective replication in permissive chicken embryo fibroblasts. In this report, we show that splicing at the src 3' splice site (3'ss) is further negatively regulated by the suppressor of src splicing cis element which is located approximately 100 nucleotides upstream of the src 3'ss. The increase in splicing at the src 3'ss results in a corresponding increase in splicing at a cryptic 5'ss within the env gene. Two classes of replication-competent revertants of the src oversplicing mutant (pSAP1) were produced after infection, and these mutants were characterized by molecular cloning and sequence analysis. Class I revertants are transformation-defective revertants in which the src 3'ss and the src gene are deleted by homologous recombination at several different sites within the imperfect direct repeat sequences that flank the src gene. Cells infected with these transformation-defective revertants produce lower levels of virus particles than cells infected with the wild-type virus. Class II revertants bear small deletions in the region containing the branchpoint sequence or polypyrimidine tract of the src 3'ss. Insertion of these mutated sequences into pSAP1 restored inefficient splicing at the src 3'ss and efficient replication in chicken embryo fibroblasts. All of these mutations caused reduced splicing at the src 3'ss when they were tested in an in vitro splicing system. These results indicate that maintenance of a weak src 3'ss is necessary for efficient Rous sarcoma virus replication.

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).

A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus.

Regulation of splicing of Rous sarcoma virus (RSV) RNA primary transcripts is necessary, as with other retroviruses, to allow for the accumulation of unspliced RNA and approximately equivalent amounts of spliced env and src mRNAs. Previous studies have indicated that the env 3' splice site is suboptimal because it has a nonconsensus branchpoint sequence and that this suboptimal splice site is required for virus replication (R. A. Katz and A. M. Skalka, 1990, Mol. Cell Biol. 10:696-704). We show in this report that the RSV src 3' splice site is also suboptimal. Mutagenesis of the src polypyrimidine-rich tract, which is interspersed with purines, to an uninterrupted 14-nt pyrimidine tract resulted in a three- to fourfold increase in the level of src splicing. Concomitant with this increase in src splicing, a cryptic 5' splice site within the env gene was activated. Splicing at this splice site is normally detected in nonpermissive mammalian cells were src splicing is elevated but occurs at low levels in permissive chicken embryo fibroblasts (CEF). In CEF, mutant viruses with the improved src 3' splice site replicated significantly slower than the wild-type virus. Transformation-defective revertants lacking the src 3' splice site were rapidly selected after passage of the chicken cells infected with the mutant virus. Thus, an inefficient src 3' splice site appears to be necessary for the efficient replication of RSV.

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.

Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) RNA follows a complex splicing pathway in which a single primary transcript either remains unspliced or is alternatively spliced to more than 30 different singly and multiply spliced mRNAs. We have used an in vitro splicing assay to identify cis elements within the viral genome that regulate HIV-1 RNA splicing. A novel splicing regulatory element (SRE) within the first tat coding exon has been detected. This element specifically inhibits splicing at the upstream 3' splice site flanking this tat exon. The element only functions when in the sense orientation and is position dependent when inserted downstream of a heterologous 3' splice site. In vivo, an HIV-1 SRE mutant demonstrated a decrease in unspliced viral RNA, increased levels of single- and double-spliced tat mRNA, and reduced levels of env and rev mRNAs. In addition to the negative cis-acting SRE, the flanking 5' splice site downstream of the first tat coding exon acts positively to increase splicing at the upstream 3' splice sites. These results are consistent with hypotheses of bridging interactions between cellular factors that bind to the 5' splice site and those that bind at the upstream 3' splice site.