JOURNAL OF VIROLOGY, June 2003, p. 6284–6292 0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.11.6284–6292.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 11
The Simian Immunodeficiency Virus 5⬘ Untranslated Leader Sequence Plays a Role in Intracellular Viral Protein Accumulation and in RNA Packaging Jignesh Patel,1 Shainn-Wei Wang,1,2 Elena Izmailova,1,2 and Anna Aldovini1,2* Department of Medicine, Children’s Hospital,1 and Department of Pediatrics, Harvard Medical School,2 Boston, Massachusetts Received 10 July 2002/Accepted 28 February 2003
We investigated the role of 5ⴕ untranslated leader sequences of simian immunodeficiency virus (SIVmac239) in RNA encapsidation and protein expression. A series of progressively longer deletion mutants was constructed with a common endpoint six nucleotides upstream of the gag initiation codon and another endpoint at the 3ⴕ end of the primer binding site (PBS). We found that efficient intracellular Gag-Pol protein accumulation required the region between the PBS and splice donor (SD) site. Marked reduction of genomic RNA packaging was observed with all the deletion mutants that involved sequences at both the 5ⴕ and at the 3ⴕ ends of the major SD site, and increased nonspecific RNA incorporation could be detected in these mutants. RNA encapsidation was affected only modestly by a deletion of 54 nucleotides at the 3ⴕ end of the SD site when the mutant construct p⌬54 was transfected alone. In contrast, the amount of p⌬54 genomic RNA incorporated into particles was reduced more than 10-fold when this mutant was cotransfected with a construct specifying an RNA molecule with a wild-type packaging signal. Therefore, we conclude that the 175 nucleotides located 5ⴕ of the gag initiation codon are critical for efficient and selective incorporation of genomic RNA into virions. This location of the SIV ⌿ element provides the means for efficient discrimination between viral genomic and spliced RNAs.
possesses a functional intron within the long terminal repeat (6, 10, 40, 43). For HIV-1, the encapsidation signal has been shown to be composed of at least four stem-loop structures distributed both at the 5⬘ and 3⬘ ends of the major SD site (1–3, 8, 14, 22, 24, 42). SIV and HIV-2 are more closely related than SIV and HIV-1. The 490-nucleotide (-nt) fragment of the SIV leader sequence, extending from the RNA cap site to a few nucleotides after the first major SD junction, is 90% identical to the HIV-2 leader. In contrast, the 55 nt that precedes the gag ATG is highly divergent in SIV and HIV-2. The precise contribution of fragments of the leader sequence to SIV and HIV-2 encapsidation is controversial. For example, a 61-nt deletion immediately 3⬘ of the SD site was shown to abolish packaging in HIV-2 (30). However, other groups reported that small deletions at the 3⬘ of the SD site had only moderate effects on HIV-2 RNA encapsidation (13, 26). Deletions of HIV-2 and SIV sequences 5⬘ of the SD site were shown to significantly affect packaging in a number of studies (13, 16, 17, 19, 26). An SIV vector including the first 424 nt of the SIV leader up to the dimerization initiation site (DIS), was shown to provide SIVmediated gene transfer at the same efficiency as a vector including a longer leader sequence that extended into the gag open reading frame (36). Critical packaging signals in sequences located at the 5⬘ end of the SD are present in spliced and nonspliced mRNAs, suggesting that additional signals in other parts of the genome, possibly in the long terminal repeat intron, are required to provide selectivity for the viral genomic RNA. It has been suggested that HIV-2 possesses a unique mechanism of RNA selection that might circumvent this problem: the newly translated Gag polyprotein binds to the pack-
The 5⬘ untranslated leader of retroviruses is involved in a variety of functions that affect different steps of the retrovirus life cycle. Among these are RNA elongation, RNA splicing, protein translation, genomic RNA dimerization and packaging, and initiation of reverse transcription (5, 9, 17, 18, 24, 27, 29). While RNA elongation, RNA splicing, and initiation of reverse transcription are associated with short and well-defined sequences, RNA translation and packaging elements are distributed over longer sequences that are less well defined in length and structure. Packaging of retroviral RNAs involves the selective encapsidation of an unspliced genomic RNA dimer into the virion (12, 34). For many retroviruses, critical portions of the packaging signal (⌿) have been mapped between the major splice donor (SD) site and the gag initiation codon (12, 34). This location of ⌿ permits discrimination between full-length genomic RNA and subgenomic RNA species during the packaging process (25, 32, 37). RNA incorporation occurs via interactions between the cis-acting elements, which are located in the 5⬘ untranslated RNA leader, and the trans-acting factors of the Gag polyprotein (1, 11, 15, 31, 35). Although human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are evolutionary related, they share little sequence homology in the noncoding regions of their genome. The limited homology is particularly evident in the 5⬘ untranslated leader sequence: SIV has a significantly longer leader sequence than HIV-1 (33) and it
* Corresponding author. Mailing address: Children’s Hospital, Enders 609, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 3558426. Fax: (617) 566-4721. E-mail:
[email protected]. 6284
VOL. 77, 2003
SIV 5⬘ UNTRANSLATED LEADER SEQUENCE AND RNA PACKAGING
FIG. 1. Schematic representation of the deletion constructs. The deleted nucleotides are indicated, and their numbering is according to the Gene Bank sequence with accession no. m33262. Positions of the PBS, major SD site, DIS, and gag ATG codon are also indicated.
aging signal in the same genomic RNA molecule that has been used for its translation (21). This mechanism has been termed cis-packaging. The 5⬘ untranslated leader sequences of several retroviruses have been shown to contain signals required for the efficient translation of the Gag polyprotein (27, 39, 41). For example, the secondary structure of the HIV-1 RNA packaging signal sequence upstream of the gag initiation codon has been shown to have an inhibitory effect on the translation of the Gag polyprotein and unwinding of this structure is required for efficient gag expression (27). Moreover, the HIV-1 gag open reading frame has been shown to contain an internal ribosome entry site (IRES) that affects translation of both Gag Pr55 and of the truncated form of Gag p40 (5). In contrast, the leader sequence of SIV contains an IRES between the SD and the start codon of gag and directs translation of the full-length Gag polyprotein (29). The secondary structure of the 5⬘ untranslated leader appears to be not important for SIV IRES function (29). To understand the role of SIV leader sequence in RNA translation and packaging in more depth, we constructed a series of deletion mutations in the sequences located between the primer binding site (PBS) and the gag initiation codon and investigated their ability to produce viral proteins and package RNA. We show that the deletion of sequences at the 5⬘ end of the first major SD site reduces the intracellular accumulation of viral Gag-Pol proteins. We further show that the deletion of sequences both at the 5⬘ and 3⬘ ends of the SD site significantly affect the rate and the specificity of RNA encapsidation. MATERIALS AND METHODS Plasmid construction. All deletion mutants are derivatives of the infectious clone pMA239 (GenBank accession no. m33262) (38). Oligonucleotide-mediated site-directed mutagenesis by overlapping extension was employed to generate DNA fragments containing the appropriate deletion and restriction sites for subsequent cloning (20). To generate p⌬54, p⌬105, p⌬175, and p⌬221 a three-part ligation was carried out for each construct, using two PCR fragments and the BamHI/AatII fragment from pMA239 that carries the plasmid backbone. The two PCR fragments, one covering sequences located between the AatII site and the 5⬘ end of the deletion, the other covering sequences between the 3⬘end of the deletion and the BamHI site (Fig. 1) were ligated with each other by using an added XmaI site, positioned, respectively, at the 3⬘ and 5⬘ ends of the two fragments. The individual deletions span the nucleotides whose numbers are indicated in Fig. 1. The nucleotide numbers reported in this figure refer to the
6285
SIVmac239 sequence with accession no. m33262. To construct p⌬89 and p⌬158 plasmids, a DNA linker containing the splice donor and two flanking XbaI sites (forward strand, 5⬘-CCGGGTTGCAGGTAAGTCGAAC-3⬘; reverse strand, 5⬘GGCCCAACGTCCATTCACGTTG-3⬘) was cloned into XmaI-digested p⌬105 and p⌬175 plasmids. All the constructs were verified by sequencing. The plasmids expressing the Tat and Rev proteins were, respectively, SIV clone P11 and SIV clone P8 (10). The plasmid p239Luc was generated by inserting the gene for p24 and the SIVmac239 leader sequence, starting from the first nucleotide of the R region (nt ⫹775) and extending to the translation initiation codon of the Gag polyprotein (nt ⫹1309 of sequence accession no. m33262) at the 3⬘ end of the SP6 promoter. These fragments were amplified by PCR as KpnI-NheI and NheI-NcoI fragments, respectively, and were cloned into NheI-NcoI-digested pSP-luc⫹NF fusion vector (Promega, Madison, Wis.). Plasmids p⌬105Luc and p⌬175Luc constructs were constructed by amplifying the fragments containing the leader sequence (SIV nt 775 to nt 1309) from the p⌬105 and p⌬175 mutant constructs, respectively, and cloning them into NheI-NcoI-digested p239Luc. Cell lines and transfection. 293T cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 g/ml), and 2 mM L-glutamine (Invitrogen, Carlsbad, Calif.). Transfections were carried out by the calcium phosphate precipitation method, using 15 g of DNA for each deletion mutant construct, 5 g of SIV clone P11 and 5 g of SIV clone P8, or 10 g of pUC19 when pMA239 and p⌬54 were used in cotransfection experiments (7). When pMA239 was cotransfected with p⌬54, cells were transfected with 10 g of DNA for each proviral construct or 10 g of p⌬54 and different amounts of pMA239 and compensatory amounts of pUC19. The percentage of transfected cells was evaluated by intracellular SIV protein staining. Western blot, ELISA, and RT assays. Supernatants from transfected 293T cells were collected after 48 h, clarified from cellular debris by spinning at 2,000 ⫻ g for 10 min, and filtered through a 0.45-m-pore-size filter. To evaluate intracellular viral protein accumulation, cells were lysed in 0.1% Triton-X and the intracellular Gag-related proteins were quantified using a p27 enzyme-linked immunosorbent assay (ELISA) kit (p27 Core Profile kit; Coulter, Brea, Calif.). Virus was pelleted from clarified supernatants through a 15% (wt/vol.) sucrose gradient spun at 18,000 ⫻ g for 3 h. Pellet samples equivalent to 15 ng of p27 (quantified by ELISA), or equal amounts of cellular protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), transferred to nitrocellulose, and probed with SIVmac251-positive serum as described previously (31). 125I-protein A (New England Nuclear) was used to detect SIV-specific proteins by autoradiography. Virus production in the supernatants was evaluated by p27 ELISA and reverse transcriptase (RT) assay. For the RT assay, 10 l of each virus supernatant was mixed with 50 l of the reaction mixture [60 mM Tris-HCl, pH 8.0; 12 mM MgCl2; 72 mM KCl; 144 nM EGTA; 432 nM glutathione; 2 mM dithiothreitol; 0.1% Triton X; oligo(dT)15, 6 g/ml; poly(A), 12 g/ml; 7.5 Ci of [32P]TTP], and the reaction was carried out at 37°C for 1 h. One-sixth of the reaction mixture was spotted on DE81 Whatman paper, air dried, and washed three times with 2⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and two times with 95% ethanol. The intensity of each dot was quantified using a Molecular Dynamics PhosphorImager. RT-PCR. To detect cellular RNA, transfected cells were lysed with Triazol reagent (Invitrogen) and RNA was extracted as suggested by the manufacturer. To avoid contamination with plasmid DNA, both cellular and particle-derived viral RNA were digested with 10 U of DNase I (Invitrogen) in the presence of 80 U of recombinant RNasin (Promega). To quantify viral RNA accumulation, approximately 100 ng of total cellular RNA was used for amplification using gag-specific primers A31 (5⬘-CAAATCCAGTGGATGTA-3⬘) and SIVc 2537 (5⬘-TGCTTTCTCTCTGCTTT-3⬘), env specific-primers SIV 934 (5⬘-CTGCCA TTTTAGAAGTA-3⬘) and SIVc 7200 (5⬘-TTGCATCTCATAGTAATGCATA ATGG-3⬘), or nef-specific primers SIV9224 (5⬘-ACCTTGCTATCGAGAGTAT ACCAG-3⬘) and SIVc 9501 (5⬘-CCTCACAAGAGAGTGAGCTCA-3⬘). -actin mRNA was amplified as a control using the following primers: actin-S (5⬘-ATG TTTGAGACCTTCAACAC-3⬘) and actin-A (5⬘-CACGTCACACTTCATGAT GG-3⬘). To detect viral RNA in particles, a virus supernatant amount equivalent to 15 ng of p27 was centrifuged and the amount of pellet particles was determined by p27 ELISA. Equal amounts of pelleted virus were resuspended in Triazol, in the presence of 30 g of yeast tRNA as a carrier to monitor final RNA recovery, and RNA was isolated. Quantitative RT-PCR was performed on samples containing equal amounts of recovered RNA, after DNase I treatment, as previously described (31). The negative controls included a sample from an RT-PCR lacking input RNA and an RT-PCR with RNA extracted from a mock-transfected supernatant. A PCR on an equivalent amount of RNA which did not undergo reverse transcription was carried out for each sample to exclude
6286
PATEL ET AL.
J. VIROL.
FIG. 2. Intracellular gene expression with SIV deletion mutants. (A) Characterization of standards. A fixed amount of total cellular RNA from 293T cells transfected with the SIV mutants was amplified by RT-PCR using four set of primers (䊐, gag; ■, nef; E, env; F, -actin). The linear range of the product amounts obtained after different numbers of cycles is shown in the graph. The linear range of the twofold RNA dilutions also shown: amplification was carried out for 22 cycles when gag primers were utilized, 18 cycles with nef primers, 28 cycles with env primers, and 18 cycles with -actin primers. (B to E) RT-PCR analysis of RNA from cells transfected with viral mutant plasmids only (B); viral mutant plasmids, the Tat-expressing plasmid p11, and the Rev-expressing plasmid p8 (C); viral mutant plasmids and p11(D); or viral mutant plasmids and p8 (E). Plasmid p11 or p8 or both were added to the transfections whose analysis is shown in lanes 3 to 7 of the different panels. Wild-type plasmid or p⌬54 were cotransfected with corresponding amounts of pUC19. For each RNA, a control PCR run in absence of RT is shown next to the RT-PCR. The panels show the results of one representative experiment. Similar results were obtained in three separate experiments. incomplete DNase I treatment. RNA samples were obtained from three independent transfections for each construct. To asses the percentage of RNA incorporation by mutant viruses relative to the parental wild type, the intensity of a bands corresponding to RNA from mutant viruses was compared with that of serially diluted of wild-type virus RNA. To avoid overamplification by PCR, we chose a number of cycles that permits to obtain standard RNA bands whose intensity is within linear range. In vitro transcription and translation assays. Plasmids p239Luc, p⌬105Luc, and p⌬175Luc were linearized by digestion with XbaI which cuts the DNA at the 3⬘ end of the luciferase gene. Ribomax Large Scale RNA Production System-SP6 (Promega) was used to carry out transcription reactions according to the manufacturer’s protocol. RNA transcripts were purified, the integrity of the RNA was verified by agarose gel electrophoresis, and the RNA concentration was measured by spectrophotometry. One microgram of RNA for each construct was translated using a nuclease-treated rabbit reticulocyte lysate (Promega), as previously described (29). Translation products were separated by SDS-PAGE, and the dried gel was subjected to autoradiography. The intensity of each band was quantified by a Molecular Dynamics PhosphorImager. In addition, translated HIV-1 p24 was measured by ELISA, and luciferase activity for each sample was measured using a luciferase assay kit (Promega).
RESULTS Transcription and translation of the mutated SIV genomic RNAs. To investigate the role of the SIV 5⬘ untranslated leader sequence in viral protein translation and RNA packaging, we constructed a series of 5⬘ leader deletion mutants (Fig. 1). Progressively larger deletions, from a minimum of 54 nt to a maximum of 221 nt, were introduced into the 5⬘ leader se-
quence of the pMA239 proviral vector, in the sequence that extends from the PBS to the gag AUG. With the exception of mutant p⌬54, no viral gene expression could be detected when the plasmids carrying the mutated proviruses were transfected alone (Fig. 2B), with a plasmid expressing Tat (Fig. 2D) or with a plasmid expressing Rev (Fig. 2E). The intracellular gag-pol mRNA levels were evaluated by RT-PCR with primers specific for the gag gene, while singly and doubly spliced mRNAs were evaluated with env- and nefspecific primers, respectively. However the gene expression defect observed in these mutants could be corrected when the SIV deletion mutant plasmids were transfected with both tat and rev cDNAs. Under these conditions, the gag-pol mRNA could be detected for all mutants in amounts comparable to the wild-type virus (Fig. 2C). Tat and Rev activities are both critical for efficient HIV-1 transcription and nuclear export. The plasmids containing the SIV tat and SIV rev cDNAs encode bona fide tat and rev mRNAs, and each of their mRNAs can be detected after transfection by RT-PCR with nef-specific primers (Fig. 2D and E), but the provirus-derived tat and rev mRNAs are undetectable when the mutants are transfected alone (Fig. 2B, nef primers). We conclude that viral gene expression was abnormal in all mutants, with the exception of p⌬54, because they lack Tat and Rev functions.
VOL. 77, 2003
SIV 5⬘ UNTRANSLATED LEADER SEQUENCE AND RNA PACKAGING
6287
TABLE 1. Quantitative analysis of SIV proteins in cell lysates and in virionsa Construct
-Gal (% of wt)
Cellular Gag (ng/mg)
Viral Gag (ng/ml)
RT activity (% of wt)
pMA239 p⌬54 p⌬89 p⌬105 p⌬158 p⌬175 p⌬221
100 115 ⫾ 3 120 ⫾ 7 89 ⫾ 3 100 ⫾ 3 120 ⫾ 3 110 ⫾ 18
265 ⫾ 10 248 ⫾ 11 97 ⫾ 5 30 ⫾ 3 15 ⫾ 5 14 ⫾ 3 11 ⫾ 3
465 ⫾ 12.5 276 ⫾ 4.6 15.1 ⫾ 0.21 3 ⫾ 0.45 2 ⫾ 0.1 1 ⫾ 0.15 2 ⫾ 0.53
100 66.5 ⫾ 3.5 6.5 ⫾ 0.71 3.5 ⫾ 0.7 3 ⫾ 0.01 2 ⫾ 1.41 4 ⫾ 0.01
a A construct expressing LacZ was cotransfected with the viral mutants. -Gal (nanograms per micrograms of total cellular proteins) activity was measured in the cell lysates. Amounts of intracellular Gag products and virus-associated Gag (nanograms per milliliter of supernatant) were measured by p27 ELISA. Virusassociated Gag and RT activity were measured on pelleted particles. The averages and standard errors of the means of values obtained in three independent experiments are reported.
The accumulation of genomic viral RNA to levels similar to the wild type after cotransfection of plasmids expressing Tat and Rev, but not in absence of one of them, is an indication of appropriate Tat and Rev function in these cells. However, singly spliced messenger RNAs, which include the env mRNA, could not be detected after transfection of tat and rev in the mutants that showed a gene expression defect. The same splicing defect that prevents Tat and Rev gene expression, observed when the mutants are transfected alone, is the most likely reason why accumulation of env mRNA does not occur in these mutants when transcription and nuclear export are efficient. While this defect was predictable for mutants p⌬105, p⌬175, and p⌬221, which lack the first major SD, it was not anticipated for p⌬89 and p⌬158. These mutants were derived from p⌬105 and p⌬175 by inserting 16 nt spanning the first SD at the deletion junction, in an attempt to correct the transcription defect in p⌬105 and p⌬175. It is possible that the sequence necessary to achieve correct splicing extends beyond the 16-ntspanning SD or that the inserted SD does not fold in the appropriate secondary structure because of the modification in the neighboring sequences. We then investigated the effects of the SIV leader sequence deletions on intracellular viral protein accumulation. Transfection efficiencies, evaluated by -galactosidase (-Gal) activity (Table 1) and by intracellular Gag staining (not shown), were highly similar for all constructs. However, the intensity of cell staining varied with different constructs, suggesting that different amounts of Gag protein might have accumulated within the cells. A significant reduction in the supernatant accumulation of Gag and RT proteins was also observed for all the mutants, with the exception of p⌬54, which showed a twofold reduction in viral protein accumulation when compared to the wild-type virus (Table 1). This reduction ranged from approximately 20-fold for p⌬89 to 50- to 200-fold for the larger deletions and paralleled a similar reduction in the intracellular accumulation of Gag-associated products (Table 1, cellular Gag and viral Gag). Cellular protein lysates from transfected 293T cells were also analyzed by Western blotting (Fig. 3A). The two assays indicate that the levels of steady-state intracellular Gag-Pol inversely correlated with the length of the 5⬘ leader sequence deletion, and that the 5⬘ SIV leader sequence may be important for efficient Gag-Pol accumulation. Furthermore, the
FIG. 3. Western blot analysis of viral protein in cell lysates and in virions. (A) Analysis of viral protein in cell lysates from transfected 293T cells. Fifty micrograms of total cellular protein lysate were loaded in each lane. (B) Protein analysis of pelleted SIV virions. Protein samples equivalent to 20 ng of Gag, evaluated by p27 ELISA, were loaded in each lane. An SIV-positive macaque serum was used to detect the SIV proteins. The detected SIV viral proteins and their molecular weight are indicated in both panels.
Western blot analysis indicates that processing was less efficient in most of the mutants and that only the p⌬54 Env protein profile was similar to that of the wild type. In contrast (and as expected in light of the absence of env mRNA), gp120 and gp41could not be detected in constructs p⌬89, p⌬105, p⌬158, p⌬175, and p⌬221. The lack of detection of RT (p66) and integrase (p30) in the cell lysates of some of the mutants is due to the lower amount of total viral proteins present in the cell lysate, as p66 and p30 can be detected for all the mutants when equal amounts of viral particle lysates are analyzed (Fig. 3B). We conclude that deletions at the 5⬘ end of the first major SD site in the SIV leader sequence affect Gag and Gag/Pol polyprotein accumulation, possibly by reducing their translation rate. Analysis of protein and RNA content of mutated viral particles. Towards our goal of investigating the effect of the SIV 5⬘ untranslated leader sequence on RNA encapsidation, we evaluated the particle protein and RNA composition by SDSPAGE and by RT-PCR. To confirm that the reduced intracellular accumulation of the Gag-Pol polyprotein did not result in
6288
PATEL ET AL.
FIG. 4. RNA content of viral particles determined by RT-PCR. RNA samples from pelleted virions calibrated to correspond to equal Gag amounts were reverse transcribed using SIV gag-specific primers to detect the genomic RNA (A), nef-specific primers to detect total viral RNA (A), or -actin to detect a representative cellular mRNA (B). (A) The linear range of the gag and nef standards (E gag, F nef) is shown. The histogram reports mutant RNA incorporation as a percentage of wild-type virus. The mean values and the standard errors of the means (error bars) derived from three independent experiments are shown.
major protein defects in the released particles, we evaluated the particle protein composition by SDS-PAGE (Fig. 3B). SIV structural Gag and Pol proteins could be detected in all mutant particles. However, the detection of higher amounts of intermediate Gag products in particle lysates (calibrated by total amount of Gag) indicates that Gag processing was less efficient in mutants ⌬89, ⌬105, ⌬158, ⌬175, and ⌬221 (Fig. 3B). As expected, since we did not detect Env proteins among intracellular viral proteins, no significant amounts of gp120 and gp41 Env proteins were observed in mutant viruses ⌬89, ⌬105, ⌬158, ⌬175, and ⌬221. Absence of Env proteins and reduced Gag and Gag-Pol processing do not affect particle production, which can be achieved with unprocessed Gag. Therefore we concluded that although some mutant virus protein profiles were different from the wild type, the detected abnormalities are unlikely to directly affect RNA packaging specificity. To evaluate whether the 5⬘ leader deletions altered viral genomic RNA packaging, we carried out RT-PCR with primers specific for genomic RNA on RNA samples derived from wild-type and mutant particles containing equivalent amounts of p27 (Fig. 4). We also estimated the specificity of RNA incorporation by comparing the amounts of genomic RNA, total viral RNA and -actin in the viral particles (Fig. 4). In mutant virus ⌬54, genomic RNA incorporation was equal to 75% of the wild type, suggesting that sequences downstream of the SD may not be critical to RNA encapsidation specificity (Fig. 4). In other mutants, the deletion of leader sequences 5⬘ and 3⬘ of the SD site had a more adverse effect on genomic
J. VIROL.
RNA incorporation into particles than the deletion in p⌬54. Mutant viruses ⌬89, ⌬105, ⌬158, ⌬175, and ⌬221 packaged only 15, 17, 5, 5, and 8%, respectively, of the amounts of genomic RNA packaged by wild-type virus (Fig. 4A). The amounts of total viral RNA incorporated by the mutants was higher than the amounts of genomic RNA; mutant viruses ⌬89, ⌬105, ⌬158, ⌬175, and ⌬221 packaged 30, 40, 20, 20, and 50% of wild-type levels, respectively. Nef primers that can detect both unspliced and spliced product were used in the evaluation of total viral RNA. This result indicates that larger amounts of tat and rev RNAs were incorporated into these mutants than in the wild-type virus, reflecting a reduced specificity of RNA packaging. Furthermore, we could detect significant amounts of -actin in all the mutants (Fig. 4B). Increased incorporation of mRNAs for housekeeping genes has been observed when a viral RNA that includes the packaging signal is not present in the intracellular RNA pool (28) and is considered an indicator of nonspecific RNA packaging. Finding higher amounts of viral spliced and cellular mRNA in the viral particle when a mutated viral genomic RNA is available suggests that the affinity of the packaging proteins for that mRNA is lower than the affinity for the wild-type viral genomic RNA, and therefore nonspecific packaging can occur simultaneously. Thus, the deletion of the 100 nt located 5⬘ of the SD site resulted in decreased viral genomic RNA encapsidation. The data presented in Fig. 4 showed that the deletion of 54 nt at the 3⬘ end of the SD resulted in a modest effect on the efficiency of viral genomic RNA incorporation. We entertained the possibility that the rate of incorporation of the p⌬54 genomic RNA could be more properly evaluated if this mutant was cotransfected with a vector encoding a wild-type RNA, thus permitting competition for encapsidation between wildtype and mutant sequences. We cotransfected the wild-type construct pMA239 and the p⌬54 mutant and measured the incorporation of genomic viral RNA into particles. When the two plasmids were transfected in equal amounts and the intracellular expression levels of the two gag RNAs were similar, the ⌬54 RNA was packaged into virions at only 8% of the level of the pMA239 RNA (Fig. 5B). This amount of incorporation is similar to the amount of incorporation observed for spliced mRNA in wild-type particles (4, 31), which usually results from nonspecific packaging that occurs during viral genomic packaging. When pMA239 and the p⌬54 mutant were transfected at different ratios and the rate of pMA239 genomic RNA incorporation was corrected for the relative abundance of this mRNA in the cytoplasm, preferential incorporation of the pMA239 RNA was observed at all plasmid ratios (Fig. 5B, lanes 2 to 5). The intracellular abundance of the pMA239 RNA had an effect on its rate of incorporation, as the preferential incorporation decreased with the decreased abundance of the pMA239 genomic RNA relative to the genomic p⌬54. These results suggest a greater affinity of the packaging proteins for the MA239 genomic RNA than for the ⌬54. Therefore, we conclude that the sequences located between the first major SIV SD site and the gag ATG are important for efficient RNA packaging. It is possible that competition experiments carried out to evaluate viral genomic RNA incorporation in the remaining mutants would have revealed an even more striking reduction than that observed in the experiments shown in Fig. 4. However, because quantitative RT-PCR is not very
VOL. 77, 2003
SIV 5⬘ UNTRANSLATED LEADER SEQUENCE AND RNA PACKAGING
6289
FIG. 6. In vitro translation of luciferase RNAs carrying the wildtype SIV leader sequence or two of its mutants, positioned as an IRES. (A) Diagram of the constructs used in the in vitro transcription and translation. (B) The histograms report the luciferase activity measured in translation reactions and show the average values and standard deviation (error bars) of luciferase activity for wild-type and mutant RNAs observed in four independent experiments. HIV-1 p24, measured by ELISA in the translation reactions carried out with RNAs with mutated SIV leader sequence, was 100% ⫾ 5% of that obtained with RNA with wild-type SIV leader sequence and luciferase activity of each RNA was corrected accordingly. 1, 2, 3, and 4 represent results obtained with twofold dilutions of translation reaction proteins used in the luciferase activity assay.
FIG. 5. RNA packaging of competing pMA239 and p⌬54 genomic RNAs. (A) Evaluation of intracellular gag mRNA accumulation and particle gag mRNA incorporation by RT-PCR. The constructs used in each transfection and the respective DNA amounts are listed at the top of the figure. The numbers below the RT-PCR bands indicate the ratio between the intensity of the pMA239 and p⌬54 bands detected in intracellular RNA (a); the ratio between the intensity of the pMA239 and the p⌬54 bands detected in the corresponding virion RNA (b); and the increase of the pMA239 gag RNA incorporation compared to the p⌬54 gag RNA incorporation, after correction by the relative amount of the intracellular gag RNAs (b:a). The image related to the cellular RNA is overexposed to show the low-intensity 355-bp band present in the last lane. The linear range of the viral standards utilized in this experiments is shown at the top of panel A. Standards for cellular RNAs, based on the 300-bp ⌬54 band, were also in the linear range (not shown). (B) The histogram reports the percentage of p⌬54 particle gag RNA incorporation (solid bars) relative to amount of pMA239 gag RNA (open bars), after correction by the relative amount of the intracellular gag RNAs obtained at the different plasmid ratios. One representative experiment (of three with similar results) is reported in this figure.
accurate when the RNA amounts are below 6% of the control, we opted not to conduct these experiments. Taken together, our data indicate that the SIV packaging sequence is distributed through the 175 nt that are deleted in mutant virus ⌬175 and are located 5⬘ of the gag ATG. In vitro translation of mutated gag-pol mRNAs. The analysis of intracellular vial protein for the mutants indicates that the SIV leader sequence contains sequences required for the effi-
cient accumulation of viral Gag-Pol proteins. The SIV sequences likely to play a role in this process could be those located between the PBS and SD, as Gag-Pol protein accumulation is more significantly reduced in mutant constructs with deletions in these sequences. Other investigators have reported that retroviral untranslated sequences are involved in the IRES function critical to efficient Gag-Pol translation. Therefore, we evaluated the effect of deletions in the 5⬘ untranslated SIV leader sequence on the translation of a heterologous bicistronic mRNA that includes the SIV leader sequence in an IRES-like configuration, positioned 5⬘ of the luciferase gene and 3⬘ of the coding sequence for HIV p24. We selected for study the leader sequences present in p⌬105 and p⌬175 because the mutant p⌬54 did not show a significant defect in intracellular protein and virion accumulation. In addition, with the exception of the inserted SD, the leader sequence of ⌬89 and ⌬158 mutant viruses is identical to the leader of ⌬105 and ⌬175, and ⌬89 and ⌬158 show a protein accumulation defect similar to ⌬105 and ⌬175. In vitro transcription and translation of p24 and luciferase genes were carried out using p⌬105Luc and p⌬175Luc plasmids and were compared to the translation efficiency with the luciferase gene from p239Luc, which contains the wild-type SIV leader sequence. The results of this experiment are reported in Fig. 6. The SDS-PAGE analysis (not shown) correlated with the quantitative HIV-1 p24 ELISA and luciferase assay. Translation of the RNA transcribed from p⌬105Luc produced amounts of p24 highly comparable to those obtained with the mRNA from p239Luc while the luciferase activity was reduced to 60% of the activity obtained with the wild-type RNA. Luciferase activity obtained from translation of the RNA from
6290
PATEL ET AL.
p⌬175Luc was only slightly lower than that obtained with the RNA from p⌬105Luc (Fig. 6B). These results were similar whether the assays were carried out with uncapped or capped mRNAs (Fig. 6B). These data indicate that the SIV 5⬘ untranslated leader can function as an IRES but that the 175 nt of this sequence that are deleted in p⌬105Luc and p⌬175Luc are only moderately important for efficient protein translation. Alternatively, it is possible that the translation carried out with the rabbit reticulocyte lysate cannot faithfully recapitulate translation in human cells. It is also possible that these effects are more evident when multiple RNAs are simultaneously present in a single cell and the abundance of the viral RNA is limited. DISCUSSION We show here that deletions in the SIV 5⬘ untranslated leader affect both intracellular viral protein accumulation and genomic RNA packaging and demonstrate that important packaging determinants are located both 5⬘ and 3⬘ of the SIV SD site. The results described here indicate that the SIV 5⬘ untranslated sequence plays a role in viral protein accumulation but do not reveal the molecular mechanism by which protein accumulation is altered in these mutants. gag-pol mRNA accumulation is comparable to the wild-type virus in these mutants. The effect of a negative regulator of gene expression, located at the 3⬘ end of the SD site and affecting transcription levels, was described for HIV-2 (13). We did not detect increased proviral transcription in any of our mutants in which the region downstream of the SD site was deleted. However, deletions in the SIV leader sequence did affect the steady state levels of structural proteins. We investigated the effects of the SIV 5⬘ leader sequence on viral protein accumulation in cells and on luciferase translation in vitro. Sequences in the region between the first SD site and the gag ATG are unlikely to affect intracellular Gag-Pol protein steady state accumulation, as the p⌬54 mutant, in which these sequences are deleted, did not show a major impairment in Gag protein accumulation. Deletions that extend 5⬘ of the SD site, however, resulted in a reduced accumulation of the Gag and Pol proteins in cell lysates and in the supernatants. As an IRES function has been suggested for the SIV leader sequence, we investigated the translation efficiencies conferred by leader sequences with deletions that extend further upstream of the SD site. The results observed with the proviral clones in tissue culture could not be explained by the results obtained when investigating the IRES function provided by deleted SIV leader sequences in vitro. It is possible that other processes beside translation, such as RNA stability, may have been affected in these viruses. However, their investigation is beyond the scope of this report. In concert with our observations with the SIV p⌬54 mutant transfected alone, a 61-nt deletion between the SD and gag start codon of HIV-2 was previously shown to produce no significant effect on viral protein production (30). However, small deletions in the SIV sequences located at the 5⬘ end of the DIS (corresponding to nt 1194 to 1200 in Fig. 1) have been reported not to affect the levels of virion accumulation in the transfection supernatants (18). These data are in apparent contradiction with our evidence that sequences at the 5⬘ end of the dimerization initiation site have a role in protein accumu-
J. VIROL.
lation. It is possible that the different size of these deletions can account for the differences in the results. Sequences at the 3⬘ end of the SD site were thought to be critical to the IRES function provided by the SIV leader sequence (29). We confirmed that the SIV leader sequence can provide IRES function. However, in our experiments, no major protein accumulation effect was observed when a similar deletion was present in the provirus, arguing that SIV protein translation is not dependent on these sequences. A 5⬘ leader deletion that extends 70 nt 5⬘ of the DIS resulted in reduced Gag accumulation in the supernatant of transfected cells in culture and only in a minor defect in protein translation in vitro. It is possible that the protein accumulation defect that we detected may be due in part to reduced translation efficiency and that other factors, such as decreased RNA stability, may contribute to the defect. We also investigated the role of the SIV 5⬘ untranslated sequence in RNA packaging. By comparing the RNA species identified in the particles produced by mutant and wild-type viruses, we found that the SIV sequences located upstream of the SD site were required for specific genomic RNA incorporation. Sequences located downstream of the SD also contribute to efficient genomic RNA packaging but their contribution is fully revealed only in competition experiments. Our data extend the functional similarities noted previously for SIV and HIV-2 to the RNA packaging sequences. In HIV-2, deletions of the sequences upstream of the SD site severely reduced RNA packaging (16, 26). In addition, deletion of nucleotides downstream of the major HIV-2 SD site also had a significant adverse effect on packaging function (16, 30). Most investigations of packaging signals have focused on the behavior of packaging mutants in the absence of the wild-type signal (13, 18, 19, 23, 26). As packaging proteins can bind to RNA in a sequence-nonspecific manner, it is possible that a mutated RNA packaging sequence, in which some but not all of the ⌿ signal is removed, can be incorporated more efficiently than cellular RNAs. Only a few reports addressed the function of specific packaging sequences in competition with the wild type (16, 30). Griffin et al. did not observe a decrease in packaging in a mutant in which 40 HIV-2 nt between the SD site and gag ATG were deleted. However, Poeschla et al. could not detect transduction of the HIV-2 Gag/Pol vector in which 61 nt between SD and gag ATG were deleted and the Gag/Pol proteins were provided by the transducing vector. This result indicates that the 61-nt deletion between SD and gag ATG was sufficient to exclude these molecules from the packaged pool. Similarly, we found that RNA incorporation for the SIV p⌬54 mutant was significantly different when this mutant was cotransfected with a wild-type SIV provirus, suggesting a difference in affinity between the two RNAs for packaging proteins. A comparison of the SIV 5⬘ leader deletion mutations studied here with the HIV-2 mutations described previously (26, 30) suggests that a short stretch of 10 well-conserved nt (SIV, ACACAAAAAA; HIV-2, ACACCAAAAA), located immediately after the SD site junction, is most likely critical to both the efficiency and specificity of both HIV-2 and SIV genomic RNA packaging (Fig. 7). This fragment is deleted in p⌬54 and in pE41 and is retained in the ⌬4 mutant described by McCann and Lever, in which the deletion affects predominantly the region of HIV-2 that is most divergent from SIV (26, 30).
VOL. 77, 2003
SIV 5⬘ UNTRANSLATED LEADER SEQUENCE AND RNA PACKAGING
6291
FIG. 7. Alignment of HIV-2 and SIV leader sequences form the RNA start site to the last nucleotide before the gag ATG.
In summary, our results indicate that sequence information important to packaging is present in sequences located both 5⬘ and 3⬘of the SD site. A packaging element that is distributed both 5⬘ and 3⬘ of the SD site provides a mechanism for discrimination between spliced and nonspliced messengers, as only nonspliced messenger RNAs retain the portion of the sequence located 3⬘ of the SD site. In light of these results, the ability to obtain wild-type levels of SIV gene transfer with a leader sequence that terminates at the DIS site is surprising (36). However, it is possible that packaging of SIV vectors into HIV-1 proteins might have affected this result. It will be interesting to investigate the transduction efficiency of SIV gene transfer vectors, packaged into SIV structural proteins, that include sequences located both 5⬘ and 3⬘of the SD site. ACKNOWLEDGMENTS This work was supported by NIH grant AI 36060. We thank Gretchen Schmelz for carrying out some preliminary experiments. We also thank Frederic Bertley and Richard Young for critical revision of the manuscript. REFERENCES 1. Aldovini, A., and R. A. Young. 1990. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J. Virol. 64:1920–1926. 2. Berkowitz, R. D., and S. P. Goff. 1994. Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein. Virology 202:233–246. 3. Berkowitz, R. D., M. L. Hammarskjold, C. Helga-Maria, D. Rekosh, and S. P. Goff. 1995. 5⬘ regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology 212:718–723. 4. Berkowitz, R. D., A. Ohagen, S. Hoglund, and S. P. Goff. 1995. Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo. J. Virol. 69:6445–6456. 5. Buck, C. B., X. Shen, M. A. Egan, T. C. Pierson, C. M. Walker, and R. F. Siliciano. 2001. The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site. J. Virol. 75:181–191. 6. Chatterjee, P., A. Garzino-Demo, P. Swinney, and S. K. Arya. 1993. Human immunodeficiency virus type 2 multiply spliced transcripts. AIDS Res. Hum. Retrovir. 9:331–335. 7. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752. 8. Clever, J., C. Sassetti, and T. G. Parslow. 1995. RNA secondary structure and binding sites for gag gene products in the 5⬘ packaging signal of human immunodeficiency virus type 1. J. Virol. 69:2101–2109. 9. Clever, J. L., D. A. Eckstein, and T. G. Parslow. 1999. Genetic dissociation of the encapsidation and reverse transcription functions in the 5⬘ R region of human immunodeficiency virus type 1. J. Virol. 73:101–109. 10. Colombini, S., S. K. Arya, M. S. Reitz, L. Jagodzinski, B. Beaver, and F. Wong-Staal. 1989. Structure of simian immunodeficiency virus regulatory genes. Proc. Natl. Acad. Sci. USA 86:4813–4817. 11. Darlix, J. L., C. Gabus, M. T. Nugeyre, F. Clavel, and F. Barre-Sinoussi. 1990. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J. Mol. Biol. 216:689–699.
12. Darlix, J. L., M. Lapadat-Tapolsky, H. de Rocquigny, and B. P. Roques. 1995. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J. Mol. Biol. 254:523–537. 13. Garzino-Demo, A., R. C. Gallo, and S. K. Arya. 1995. Human immunodeficiency virus type 2 (HIV-2): packaging signal and associated negative regulatory element. Hum. Gene Ther. 6:177–184. 14. Geigenmuller, U., and M. L. Linial. 1996. Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5⬘ HIV-1 genomic RNA sequence. J. Virol. 70:667–671. 15. Gorelick, R. J., D. J. Chabot, D. E. Ott, T. D. Gagliardi, A. Rein, L. E. Henderson, and L. O. Arthur. 1996. Genetic analysis of the zinc finger in the Moloney murine leukemia virus nucleocapsid domain: replacement of zinccoordinating residues with other zinc-coordinating residues yields noninfectious particles containing genomic RNA. J. Virol. 70:2593–2597. 16. Griffin, S. D., J. F. Allen, and A. M. Lever. 2001. The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity. J. Virol. 75:12058–12069. 17. Guan, Y., K. Diallo, J. B. Whitney, C. Liang, and M. A. Wainberg. 2001. An intact U5-leader stem is important for efficient replication of simian immunodeficiency virus. J. Virol. 75:11924–11929. 18. Guan, Y., J. B. Whitney, K. Diallo, and M. A. Wainberg. 2000. Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus. J. Virol. 74:8854–8860. 19. Guan, Y., J. B. Whitney, C. Liang, and M. A. Wainberg. 2001. Novel, live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences. J. Virol. 75:2776–2785. 20. Horton, R. M. S., N. Ho, J. K. Pullen, H. D. Hunt, Z. Cai, and L. R. Pease (ed.). 1993. Gene splicing by overlap extension, vol. 217. Academic Press, San Diego, Calif. 21. Kaye, J. F., and A. M. Lever. 1999. Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J. Virol. 73:3023–3031. 22. Lever, A., H. Gottlinger, W. Haseltine, and J. Sodroski. 1989. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J. Virol. 63:4085–4087. 23. Liang, C., L. Rong, R. S. Russell, and M. A. Wainberg. 2000. Deletion mutagenesis downstream of the 5⬘ long terminal repeat of human immunodeficiency virus type 1 is compensated for by point mutations in both the U5 region and gag gene. J. Virol. 74:6251–6261. 24. McBride, M. S., and A. T. Panganiban. 1996. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J. Virol. 70:2963–2973. 25. McBride, M. S., M. D. Schwartz, and A. T. Panganiban. 1997. Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J. Virol. 71: 4544–4554. 26. McCann, E. M., and A. M. Lever. 1997. Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2. J. Virol. 71:4133–4137. 27. Miele, G., A. Mouland, G. P. Harrison, E. Cohen, and A. M. Lever. 1996. The human immunodeficiency virus type 1 5⬘ packaging signal structure affects translation but does not function as an internal ribosome entry site structure. J. Virol. 70:944–951. 28. Muriaux, D., J. Mirro, D. Harvin, and A. Rein. 2001. RNA is a structural element in retrovirus particles. Proc. Natl. Acad. Sci. USA 98:5246–5251. 29. Ohlmann, T., M. Lopez-Lastra, and J. L. Darlix. 2000. An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA. J. Biol. Chem. 275:11899–11906. 30. Poeschla, E., J. Gilbert, X. Li, S. Huang, A. Ho, and F. Wong-Staal. 1998. Identification of a human immunodeficiency virus type 2 (HIV-2) encapsi-
6292
31. 32. 33. 34. 35. 36. 37.
PATEL ET AL.
dation determinant and transduction of nondividing human cells by HIV-2based lentivirus vectors. J. Virol. 72:6527–6536. Poon, D. T., G. Li, and A. Aldovini. 1998. Nucleocapsid and matrix protein contributions to selective human immunodeficiency virus type 1 genomic RNA packaging. J. Virol. 72:1983–1993. Prats, A. C., C. Roy, P. A. Wang, M. Erard, V. Housset, C. Gabus, C. Paoletti, and J. L. Darlix. 1990. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J. Virol. 64:774–783. Regier, D. A., and R. C. Desrosiers. 1990. The complete nucleotide sequence of a pathogenic molecular clone of simian immunodeficiency virus. AIDS Res. Hum. Retrovir. 6:1221–1231. Rein, A. 1994. Retroviral RNA packaging: a review. Arch. Virol. Suppl. 9:513–522. Rein, A., D. P. Harvin, J. Mirro, S. M. Ernst, and R. J. Gorelick. 1994. Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus. J. Virol. 68:6124–6129. Rizvi, T. A., and A. T. Panganiban. 1993. Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. J. Virol. 67:2681–2688. Sakaguchi, K., N. Zambrano, E. T. Baldwin, B. A. Shapiro, J. W. Erickson, J. G. Omichinski, G. M. Clore, A. M. Gronenborn, and E. Appella. 1993. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. Proc. Natl. Acad. Sci. USA 90:5219–5223.
J. VIROL. 38. Shibata, R., M. Kawamura, H. Sakai, M. Hayami, A. Ishimoto, and A. Adachi. 1991. Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells. J. Virol. 65:3514–3520. 39. Sonstegard, T. S., and P. B. Hackett. 1996. Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag. J. Virol. 70:6642– 6652. 40. Unger, R. E., M. W. Stout, and P. A. Luciw. 1991. Simian immunodeficiency virus (SIVmac) exhibits complex splicing for tat, rev, and env mRNA. Virology 182:177–185. 41. Vagner, S., A. Waysbort, M. Marenda, M. C. Gensac, F. Amalric, and A. C. Prats. 1995. Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/ PTB splicing factor. J. Biol. Chem. 270:20376–20383. 42. Vicenzi, E., D. S. Dimitrov, A. Engelman, T. S. Migone, D. F. Purcell, J. Leonard, G. Englund, and M. A. Martin. 1994. An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. J. Virol. 68:7879– 7890. 43. Viglianti, G. A., P. L. Sharma, and J. I. Mullins. 1990. Simian immunodeficiency virus displays complex patterns of RNA splicing. J. Virol. 64:4207– 4216.
