Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.

HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3' splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5' splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3' splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.

Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.

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.

Gag-processing defect of human immunodeficiency virus type 1 integrase E246 and G247 mutants is caused by activation of an overlapping 5' splice site.

We have previously described several human immunodeficiency virus type 1 (HIV-1) mutants that are characterized by an excessive-RNA-splicing phenotype and reduced virus particle production. In one of these mutants (NLD2up), the sequence of 5' splice site D2 was changed to a consensus splice donor site. This splice site overlaps the HIV-1 integrase reading frame, and thus, the NLD2up mutant also bears a G-to-W change at amino acid 247 of the integrase. A previously described E-to-K mutant at position 246 of the C-terminal domain of the integrase, which resulted in a G-to-A mutation at the +3 position of overlapping splice donor D2 (NLD2A3), was also shown to affect virus particle production and Gag protein processing. By using second-site mutations to revert the excessive-splicing phenotype, we show that the effects on Gag protein processing and virus particle production of both the NLD2up and NLD2A3 mutants are caused by excessive viral RNA splicing due to the activation of the overlapping 5' splice site and not to the changes in the integrase protein. Both integrase protein mutations, however, are lethal for virus infectivity. These studies suggest that changes in the usage of overlapping splice sites may be a possible alternative explanation for a defective virus phenotype resulting from changes in protein-coding sequences or in the nucleotide sequence during codon optimization.

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages.

BACKGROUND: Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication. RESULTS: Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. CONCLUSION: While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals.

Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA.

To distinguish the inhibitory effect of anti-sense RNA on translation from the effect on splicing, a plasmid (pLC32) was constructed from a cDNA clone of the Rous sarcoma virus (RSV) envelope gene (env) mRNA. Transcription of this plasmid results in the synthesis of RNA identical to the RSV env gene mRNA which does not require splicing to be expressed. Plasmids derived from pLC32 were also constructed in which the env gene coding sequence and 5' noncoding leader sequences were inserted in the opposite orientation relative to the RSV long terminal repeats (LTRs). pLC32 DNA transfected by the calcium phosphate coprecipitation technique efficiently rescued infectious virus from quail cells infected with an RSV mutant deleted in the env gene [R(-)Q cells], indicating that the intron sequences are dispensable in env gene expression. When the inverted constructs were cotransfected with pLC32, significantly less infectious virus was produced. The extent of the inhibition depended upon the concentration ratio of the two plasmids. The maximum inhibition (80%) occurred when the ratio of inverted constructs to pLC32 was 12:1. The inhibition is specific for the inverted orientation since cotransfection of pLC32 with several other plasmids containing viral LTRs and defective src and env genes at similar concentrations did not inhibit the production of infectious virus. In addition, the inverted constructs did not interfere with the expression of an LTR-driven chloramphenicol acetyltransferase gene. When cotransfected with a wild-type Prague A RSV DNA plasmid (pJD100), the inverted constructs also greatly inhibited expression and replication of virus in R(-)Q quail cells. These data suggest that the specific inhibition is caused by hybridization of complementary RNA transcribed from the inverted constructs to the env mRNA, thereby blocking its expression. The fact that expression of both intron-containing and intronless clones are inhibited to the same extent suggest that inhibition by anti-sense RNA from the env exon regions does not act at the level of RNA splicing.

Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.

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.

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.

Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts.

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication 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.

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.

Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing.

In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.

BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.

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.

Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning.

The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement.

MHV nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand MHV RNA.

We have found that genomic RNA synthesis is inhibited by cycloheximide in cells infected with mouse hepatitis virus, strain A59 (MHV-A59), in agreement with previously published results (Sawicki, S.G. and Sawicki, D.L. (1986) J. Virol, 57, 328-334). In the present study, the fate of the residual genomic RNA synthesized in the presence of cycloheximide was determined. Nearly all of the genomic RNA synthesized in the presence of drug was incorporated into nucleocapsid structures, suggesting that even in the absence of protein synthesis, genomic RNA synthesis and encapsidation are coupled in MHV-infected cells. Sufficient free nucleocapsid N protein was available for this purpose, since the pool of soluble N protein was determined to decay with a half-life of approximately one hour. Negative strand RNA is the template for the synthesis of both genomic and subgenomic positive strand RNA, and would be predicted to accumulate primarily during the early phases of the lytic cycle. In agreement with this prediction, negative strand RNA accumulated during the first 5-6 h of infection, with little additional accumulation occurring over the next 2.5 h. In marked contrast, positive strand RNA increased 5-6-fold over the same 2.5 h period. These results, taken in conjunction with published data, suggest that negative strand RNA is synthesized during the early period of the infectious cycle and is stable in infected cells and also suggest that treatment with cycloheximide at late times does not inhibit positive strand RNA synthesis indirectly by blocking the formation of negative strand templates.

Transcriptional and posttranscriptional regulation of HIV-1 gene expression.

Control of HIV-1 gene expression depends on two viral regulatory proteins, Tat and Rev. Tat stimulates transcription elongation by directing the cellular transcriptional elongation factor P-TEFb to nascent RNA polymerases. Rev is required for the transport from the nucleus to the cytoplasm of the unspliced and incompletely spliced mRNAs that encode the structural proteins of the virus. Molecular studies of both proteins have revealed how they interact with the cellular machinery to control transcription from the viral LTR and regulate the levels of spliced and unspliced mRNAs. The regulatory feedback mechanisms driven by HIV-1 Tat and Rev ensure that HIV-1 transcription proceeds through distinct phases. In cells that are not fully activated, limiting levels of Tat and Rev act as potent blocks to premature virus production.

Chapter 1. Regulation of HIV-1 alternative RNA splicing and its role in virus replication.

Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNA species, both completely and incompletely spliced, are produced by alternative splicing of the primary viral RNA transcript. In addition, about half of the viral RNA remains unspliced and is transported to the cytoplasm where it is used both as mRNA and as genomic RNA. In general, the identities of the completely and incompletely spliced HIV-1 mRNA species are determined by the proximity of the open reading frames to the 5'-end of the mRNAs. The relative abundance of the mRNAs encoding the HIV-1 gene products is determined by the frequency of splicing at the different alternative 3'-splice sites. This chapter will highlight studies showing how HIV-1 uses exon definition to control the level of splicing at each of its 3'-splice sites through a combination of positively acting exonic splicing enhancer (ESE) elements, negatively acting exonic and intronic splicing silencer elements (ESS and ISS elements, respectively), and the 5'-splice sites of the regulated exons. Each of these splicing elements represent binding sites for cellular factors whose levels in the infected cell can determine the dominance of the positive or negative elements on HIV-1 alternative splicing. Both mutations of HIV-1 splicing elements and overexpression or inhibition of cellular splicing factors that bind to these elements have been used to show that disruption of regulated splicing inhibits HIV-1 replication. These studies have provided strong rationale for the investigation and development of antiviral drugs that specifically inhibit HIV-1 RNA splicing.