Vol. 65, No. 8
JOURNAL OF VIROLOGY, Aug. 1991, p. 4502-4507 0022-538X/91/084502-06$02.00/0 Copyright C 1991, American Society for Microbiology
Independent Fluctuation of Human Immunodeficiency Virus Type 1 rev and gp4l Quasispecies In Vivo LIVIA PEDROZA MARTINS,' NICOLE CHENCINER,1 BIRGITTA ASJO,2 ANDREAS MEYERHANS,"3 AND SIMON WAIN-HOBSON'*
Laboratoire de Retrovirologie Moleculaire, Institut Pasteur, 75724 Paris, France'; Department of Virology, Karolinska Institute, S-10521 Stockholm, Sweden2; and Department of Virology, Institut for Medical Microbiology and Hygiene, University of Freiburg, D-7800 Freiburg, Germany3 Received 4 March 1991/Accepted 15 May 1991
The human immunodeficiency virus type 1 (HIV-1) overlapping rev and env coding sequences have been examined from sequential peripheral blood mononuclear cell DNA samples from one individual. These were the same DNA samples from which sequence data for the tat and nef/long terminal repeat loci have been derived and span a 4-year period. The rev/env sequences were established by sequencing cloned polymerase chain reaction products. The structure of the populations of rev protein sequences increased in complexity with disease, while those of the corresponding env sequences remained complex. This suggests that the rev and env populations evolved differently, probably reflecting different selection pressures. No defective rev variants encoded substitutions in residues 76 through 79, indicating that the experimental finding of down regulation of rev activity by competitive inhibition may not necessarily occur in vivo. After having analyzed three HIV loci (15% of the genome) from the same individual over 4 years, it is clear that no two loci evolved similarly, indicating the difficulties in comparing data from different loci.
while inactivating rev function, resulted in mutants capable of competitively inhibiting the native rev protein (14). In the present study, two questions were addressed. First, since this exon overlaps completely the gp4l region of the env ORF and the second tat exon, could the amino acid sequences evolve differently? Second, could defective rev gene products (14) be used as a means of autoregulation of HIV, as has been shown experimentally? Consequently, a longitudinal structure-function study of rev quasispecies in vivo as well as an ex vivo isolate was undertaken with the same DNA samples from the Swedish patient. The clinical and immunological data associated with the blood samples (Li, L2, L3, L5, and L6) and virus isolation (V6) have been previously described (3, 16). Suffice it to say that Li, L2, L3, L5, and L6 were taken in June 1985, March 1986, June 1986, February 1989, and June 1989, respectively. V6 corresponds to the HIV-1 isolate derived from cocultivation of the L6 sample with donor peripheral blood mononuclear cells. Total DNA was extracted as previously described (3, 16). The segment of the HIV-1 genome amplified from patient DNA is shown in Fig. 1A. It was decided not to amplify the 3-kb fragment encoding the two rev coding exons for three reasons. First, natural recombination between HIV-1 genomes will probably uncouple any possible cis evolution of the two exons (3, 11). Second, it was important to avoid polymerase chain reaction (PCR)-mediated recombination (17). This was shown to be sequence specific. The probability of generating PCR-mediated recombinants would surely have been greater upon amplification of the 3-kb fragment, as opposed to the 300-bp test fragments (17). Third, the first rev coding exon quasispecies were already established as part of the preceeding study of the tat gene and already shown to be reasonably homogeneous. Thus, the second exon of the rev gene was amplified as part of a larger 411-bp fragment from patient DNA by using primers REV3 and REV5 (Fig. 1A). The DNA plus strand primer REV3 (5'-CACCTCGAGTGATAGTAGGAGGCTT
RNA viruses have been described as populations of viral quasispecies (5), waiting to be selected (4). This feature is rooted in the elevated viral polymerase nucleotide misincorporation rate. Reassortment and recombination can contribute significantly to the heterogeneity of an isolate (11). The quasispecies nature of human immunodeficiency virus (HIV) in vivo and ex vivo has been intensively examined (6, 8, 16, 22, 24). Sequence analyses have described the evolution of HIV proviral sequences in vivo, thus overcoming the selective constraints of ex vivo culture upon the quasispecies (3, 16, 27). A longitudinal study of the HIV-1 proviral DNA sequences from a Swedish patient has been undertaken. Thus far, the tat and nefllong terminal repeat (LTR) loci have been analysed for the period June 1985 to June 1989 (3, 16). Disease progression was not correlated with the appearance of more efficient tat or LTR sequences. These and other studies have revealed considerable proportions of functionally defective genomes (8, 16). Here, we have focused on the second rev coding exon of HIV-1. The rev protein is a small molecule of 116 residues encoded by two exons specifying residues 1 through 26 and 27 through 116 (25). It is essential to viral replication and is involved in directing high-molecular-weight (i.e., gaglpol and env) mRNAs to the cytoplasm either by aiding transport (15) or by modulation of splicing (1, 13). The rev protein acts via a complex RNA secondary structure called the revresponsive element located in the env open reading frame (ORF) (10, 15). At least three important functional regions of the rev gene have been identified. The first is the amino terminus, which is thought to be involved in binding to the rev-responsive element (14, 19). The second is a highly basic sequence (residues 35 through 50), which is involved in targeting the rev protein to the nucleus or nucleolus (14, 19). Substitutions in the third region (residues 76 through 79), genomes, or
*
Corresponding author. 4502
VOL. 65, 1991
NOTES
L---L.
-
Il
I LTR
vI
Da
4503
EJEJE~~.rev.SI pi0 env u I%a env I I i
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A
PBMC DNA
-_
SA
TAG REV 5
SA
TAG
REV 3
5
HIV-1 LAI ATG
SD
REV IB
C
EXPRESSION VECTOR
SV40 E
REV2
REV 4
REV 3
Hindill
L===;
Sacl Bgill/BamHI
Xho
HUV I-Oly A I
ATG
SD
SA
TAG
FIG. 1. PCR amplification of the HIV-1 rev gene and construction of chimeric rev expression vector. The organization of the HIV-1 genome is shown at the top of the figure. (A) Segment (stippled box) amplified from fresh peripheral blood mononuclear cell DNA (Li, L3, L6) and from the V6 isolate by using primers REV3 and REV5. The amplified region encoded both second coding exons of rev and tat and a fragment of the gp4l gene. (B) The regions harboring the first and the second coding exons of rev (open boxes) were amplified from HIV-1 Lai plasmid DNA by using PCR primers REV1B, REV2, REV3, and REV4. The major rev splice donor and acceptor sequences are indicated by SD and SA, respectively. ATG and TAG indicate the rev gene start and stop codon. (C) The amplified fragments of HIV-1 Lai were cloned into the pASA7 expression vector containing the simian virus 40 early promoter and the hepatitis B virus poly(A) segment. The restriction sites indicated were those used in its construction and were introduced into the PCR primers. DNA fragments corresponding to rev variants (stippled box) were then subcloned by replacement of the corresponding HIV-1 Lai fragment.
GG) and minus strand primer REV5 (5'-CCGAGCTCTATA ACCCTATCTGTCC) mapped to positions 7880 to 7897 and 8308 to 8291, respectively, on the HIV-1 Lai sequence (28). In light of recent findings, LAV has been renamed HIV-1 Lai (29). The primers carried XhoI and SacI restriction sites (underlined), respectively. Optimized PCR conditions for these primers were 1.25 mM MgCl2, 25 pmol of each primer, and 2.5 U of Taq polymerase per 100-,I reaction. The DNA was amplified for 42 cycles. Thermal cycling parameters were as follows: denaturation, 95°C, 30 s; annealing, 50°C, 25 s; and elongation, 72°C, 2 min. Amplification was followed by a final 10-min step at 72°C. High-temperature Taq addition was employed. PCR products were cloned as usual (3). Plaques were screened by hybridization with a revspecific 32P-radiolabelled probe. Twenty M13 recombinants were picked from each sample and grown up for sequencing. Single-stranded DNA was sequenced by the dideoxy chain termination method with fluorescent M13 universal primers, 7-azadGTP, and Taq polymerase. The products were resolved by an Applied Biosystems 370A DNA sequencer. The error associated with PCR amplification is such that 1 to 1.5 amino acid substitutions per 20 sequences may be due to Taq polymerase. rev quasispecies. The populations of rev amino acid sequences derived from samples Li, L3, and L6, as well as the V6 ex vivo sample, are shown in Fig. 2. One sequence, identified by the symbol *, was common to all four quasispecies (L102R, L304R, L602R, and V624R), while two other sequences were common to Li and L3 only. That a major form persisted for 4 years is unusual. Previously, the major form was invariably derived from a minor form present in the preceding quasispecies (3, 16). The structure
of the population of Li rev sequences was most remarkable, being composed of three major (i.e., >5%) forms unaccompanied by any minor form, at least given the resolution of this study. The number of variants in the quasispecies appeared to increase from Li (3 sequences) to L3 (9 sequences) to L6 (13 sequences). The internal rev sequence variation for each of the three quasispecies was between 1 and 5%. The isolation of HIV resulted in the outgrowth of a minor form and homogenization of the quasispecies, as evidenced by comparing the L6 and V6 data. Relatively few defective rev sequences were identified in this exon. They included L603R (W45--R45) and L302R. The latter encoded a substitution in the major rev splice acceptor (i.e., AG/
AC-*AA/AC). The previous study of the tat sequences from the same patient (16, 18) naturally included sequence data for the first rev coding exon. The corresponding populations of protein sequences specified by this exon are shown in Fig. 3. A single form was dominant in all the samples. Noteworthy was the L5 quasispecies, in which a full 30% of sequences encoded a defective rev sequence due to a G->A transition in the initiator methionine codon. The rev splice acceptors and donor sequences 5' and 3' to this coding region were conserved (23). env quasispecies. The second rev coding exon is overlapped by part of the env ORF corresponding to the cytoplasmic tail of the gp4l transmembrane protein (Fig. 1). Consequently the nucleotide sequence of this region might be more constrained than any of the other regions examined to date. Analysis of the same nucleotide sequence, but this time in the gp4l ORF, yielded a very different profile. Thus the Li, L3, and L6 env quasispecies were all complex, being
4504
J. VIROL.
NOTES
Freq. Rel.Act 45 55 75 65 85 95 105 115 DPPPSPEGTR QARRNRRRRW RERQRQIRSI SGWIISTYLG RPAEPVPLQL PPLERLTLDC SEDCGTSGTQ GVGSPQILVE SPTVLESGTK
35
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FIG. 2. Genetic and functional complexity of rev quasispecies. The
...
sequences,
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20 1.3 20 1.4 15 1.3 ....A. 5 0.04 ....A. 5 N.D ....... 5 3.0 5 0.9 ..... 2.0 5 5 1.7 1.0 5 1.8 5 A A A A 5 0.8 .
......
aligned with respect
to the most abundant sequence in the initial sample Li. Only the differences were scored, a dot indicating sequence identity; § indicates a G-*A transition which abolished the main splice acceptor. The four populations correspond to samples Li, L3, L6, and V6. The symbols (* 0 @0 *) on the left identify the same protein sequence in different samples. The activities of 21 of 23 rev variants were determined in a transient CAT assay. N. D., not determined. CAT activities relative to HIV-1 Lai are shown in the last column. They represent the means of at least three experiments, except for L610, L614, L615, and L621, which represent the means of two experiments. CAT activities after cotransfection of pIllIAR and HIV-1 Lai homologous plasmids were typically between 10 to 20%, whereas the negative controls were of the order of 1 to 2%. Li
made up of 16, 13, and 13 sequences, respectively, none of which exceeded 20% (Fig. 4). Only three sequences were common to Li and L3. None of the 13 distinct L6 sequences were found in the preceeding Li or L3 samples. The internal env sequence variation for each of the three quasispecies was between 1 and 5%. Finally the same locus encoded 30 amino acid residues corresponding to the second tat coding exon. Despite its small size, fluctuations in the populations of sequences were observed (data not shown). This contrasts with the striking stability and homogeneity of the data from the first rev coding exon (Fig. 3). A number of features in the data presented above might suggest that stronger selection pressures were operative on the rev than on the gp41 env locus. First, in both rev exons a major form persisted for 4 years. Second, Li rev quasispecies was much more homogeneous than the corresponding env quasispecies, and finally, the complexity of the rev
MAGRSGDSDE ELLRTVRLIK FLYQS 80%
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quasispecies increased during disease progression.
This study represents the extension of analyses of the HIV-1 regulatory gene sequences within a Swedish HIV-1seropositive patient. The preceeding studies described the tat (16) and neftLTR loci (3). Figure 5 attempts to describe the evolution of HIV-1 quasispecies with time. In fact, it represents a simplification, since the notion of a quasispecies must describe not only the number and frequencies of the component sequences but also the sequence variation. Only the rev quasispecies increased in complexity during disease progression. Every locus evolved differently, and within each locus, every ORF evolved independently. In view of this and the fact that the tat, revlenv and neflLTR repre-
V6
80%
........ ..........
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FIG. 3. Sequence variability of the rev amino terminus. The nucleotide sequence data for Li, L2, and L3 have already been published (16). L5 (February 1989), L6 (June 1989), and V6 (virus derived from sample L6) samples corresponded to subsequent time points (18). rev protein sequences, given here in the one-letter code, were aligned to the dominant form present in Li. Dots indicates sequence identity. The frequency of each sequence is given to the right of the figure. Symbols (- A *) indicate identical protein sequences.
.
VOL. 65, 1991
..
NOTES
4505
815 746 806 736 77 6 726 796 786 7 56 76 6 YSPLSFQTRL PAPRGPDRPD GTEEEGGERD RIDRSGRLVDG LLAL IWDDLR SLCLFSYHRL RDLLSIAARI VELLGRRGWE VLKYWWNLLQ YWSQELKNR I..... L106E . ........
L105E
L108E L11OE L107E L102E L103E L104E L109E L112E L114E LllSE L117E L118E L121E L137E
L310E L321E L308E L323E L311E L302E L303E L304E L305E L314E L315E L319E L324E
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I......... ..R..... I......... I......... ..R ...... I......... I......... I......... I......... ..R ...... I......... I......... ..R ...... ..
E.
.........
.........
.........
20% 15% 15% 5% 5% 5% 5% 5% 5% 5% 5%
5% 5%
FIG. 4. Sequence complexity of env quasispecies in vivo. The sequences, given here in the one-letter code, were aligned with respect to the most abundant protein sequence in the initial sample, Li. Only the differences were scored, a dot indicating sequence identity. The three populations correspond to samples Li, L3, and L6. The frequency of a given protein sequence is shown on the right. The symbols (*- V) on the right identify the same protein sequence in different samples.
sented together only 15% of the HIV genome, we feel that it is not prudent to extrapolate from these data to other regions of the genome. rev function. Several studies have shown that certain segments of the HIV-1 genome harbor up to 15% of defective genomes (8, 16). Clearly, as evidenced by the high frequency of rev mutant sequences in the L5 sample, the proportion can be even greater. The contribution of defective genomes to the overall replication of HIV-1 has been addressed only in the context of particular experimental systems (14, 26). In order to test the activity of the rev variants, an expression vector was first constructed from the two coding exons of HIV-1 Lai. The backbone of the vector was derived from pASA7 (2), including the simian virus 40 early promoter and the hepatitis B virus poly(A) sequences. The first rev coding exon was amplified from the HIV-1 Lai clone by using the REVlB and REV2 pair of primers (Fig. 1B). Their sequences and coordinates were: REV1B, 5'-CCGAAGCTIT GAAATGGCTGGAAGAAGCG, positions 5548 to 5566, and REV2, 5'-CACCTCGAGACCACACAACTATTGC, positions 5710 to 5694. Furthermore, the primers encoded HindIII and XhoI restriction sites, respectively (underlined). In order to ensure efficient translation of the transcribed rev
the nonoptimal Kozak sequence (12), surrounding the natural initiator methionine codon (ATG), was changed from CCTATGG to GAAATGG by incorporation of this latter sequence into the REVlB primer (shown in boldface). The vpulenv splice acceptor sequence mapped within the REVlB primer sequence (23). To eliminate this sequence, the allimportant AG dinucleotide was substituted in the primer by a TG (also in boldface), without changing the rev coding sequence. The second coding exon of HIV-1 Lai was amplified by using the REV3 and REV4 primer pair (Fig. 1B). The sequence of REV4 was 5'-CCAGATCTGAGCTCTATAAC CCTATCTGTC, and it mapped to 8306 to 8291. It included both BglII (underlined) and Sacl (doubly underlined) restriction sites at its 5' end. Approximately 100 ng of HIV-1 Lai plasmid DNA was amplified for 15 cycles. The two amplified fragments were cloned into the HindlIl and BamHI sites of pASA7 (Fig. 1C). Eighty bases 3' to the rev splice donor and 93 bases 5' to the splice acceptor were included, so as to ensure correct splicing. This construct was designated pASArev. DNA fragments from 21 of the rev variants were subcloned into the pASArev vector. These subclones therefore represented chimeras of the first coding exon of HIV-1 Lai rev and the second rev exon from the Swedish patient
gene,
J. VIROL.
NOTES
4506
201 nelI
(206 aa)
41(99 aa) U)
v2(91 aa)
ut
r.
10-i 2(30 aa)
0
7
6 z
t1(72 aa)
Li
0
L2 L3 10
20
30
rl(25 aa) L5 L6 50 40
Time/months
FIG. 5. Complexity of different segments of t the HIV genome during disease progression. The number of dis tinct protein sequences per locus analyzed (i.e., tat, revlenv o r neflLTR) as a function of time (months) is given. Thus, rev 1 and tat 1 are deduced from the same locus (16, 18), as are rev 2, tat 2 and gp41 (this study), and nef (3). Because the lengths of the protei tn sequences are different, the number of residues is indicated i n parentheses to facilitate comparisons.
(Fig. 1C). The subclones were verified by sequencing the plasmid DNA. Rev activity was determined using pIlAlR, a rev-dependent chloramphenicol acetyltransferase (C)AT) expression deTreviouslsde plasmid (21) and Jurkat-tat T cells (20), as previously scribed. For each assay, approximately 4 x 106 cells were cotransfected with 3 ,ug of pIIIAR and 10 ,ug of recombinant rev plasmid by using the DEAE-dextran r nethod (7). All experiments included pASArev as a positive control and two negative controls: pSV2gpt and a derivatiNve of pASArev which carried a + 1 frameshift mutation in the first rev coding exon. Cells were harvested 48 h posttransfecction and lysed. CAT assays were conducted as previously described (9) after having normalized lysates to equal prc tein concentration (by using the Bio-Rad protein assay). The activities of the heterologous rev prod [ucts, relative to that of the homologous HIV-1 Lai construc St, are given on the right of Fig. 2. The values given were tthe means of at least two independent transfections, and the y represent the percentage of acetylation given by a rev vairiant relative to that of HIV-1 Lai, i.e., plasmid pASArev. [he activities of all the major forms were comparable to that c f the pASArev. Site were Variants lacking the serine (S99) phosphory ,lation found in all samples (Fig. 2), and their a(ctivity was not affected, confirming previous findings that rphosphorylation is not essential to rev activity (14). Of the n uinor forms, the activity of L603R (W45-*R45) was in th e range of the negative control, while that of the L302R variant, which lacked the splice acceptor sequence, was gireatly impaired. Site-directed mutagenesis of the third domzain (residues 76 through 79) resulted in defective rev prote ins which were able, nonetheless, to function as trans-domin iant inhibitors of the native rev protein (14, 19). In the present study, only one variant, V606R, encoded a substitution (R8 0-i>G80) in this domain which, however, did not alter rev activity significantly. The defective rev genes identified he]re (Fig. 2 and 3)
AtionhsitewSrev
antudy,inhii
ofe
encoded substitutions in the other domains. This would indicate that down regulation of rev activity by competitive inhibition has not been seized upon by HIV-1 in vivo as a possible mode of autoregulation. All the major forms tested, whether the gene were rev or tat or the sequences were from the LTR, had activities comparable to that of the corresponding HIV-1 Lai gene or sequence. However, it is possible that our experimental approaches, i.e., transfection of mammalian cells and transient expression of reporter genes such as CAT or secreted alkaline phosphatase, may not be sufficiently sensitive to detect subtle differences in gene function. This could be overcome by cloning the variants back into an infectious molecular clone. Any subtle fitness advantage of a particular variant with respect to its parent might be evident only after multiple rounds of replication. Here a problem arises. Which infectious molecular clone should be used? Should it be one of the molecular clones derived from virus adapted to established cell lines? When taken together, the three studies of HIV-1 regulatory genes and sequences derived from the patient suggest that despite the disease stage, isolation of HIV-1 always resulted in the outgrowth of a minor form in vivo. Perhaps
the single most important problem to be solved is the development of culture techniques which deform as little as possible the HIV-1 quasispecies. Until this is achieved, the quasispecies nature of HIV-1 will almost certainly constitute an important barrier to a detailed molecular definition of HIV-associated pathogenesis.
We thank Rdmi Cheynier and John Sninsky for help and discussions and Craig Rosen for the pIIIAR plasmid. L.P.M. was supported by a fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. This work was supported by grants from the Institut Pasteur, de Recherches sur le SIDA, and the Swedish l'Agence Nationale Medical Research Council.
REFERENCES 1. Chang, D. D., and P. A. Sharp. 1989. Regulation of HIV rev depends upon recognition of splice sites. Cell 59:789-795. 2. Chenciner, N., F. Delpeyroux, N. Israel, M. Lambert, A. Lim, R. Streeck, and J. F. Houssais. 1990. Enhancement of gene expression by somatic hybridization with primary cells: high-level in monkey Vero cells B surface antigen hepatitishepatocytes. of theprimary synthesis Bio/Technology 8:858-862. by fusion with 3. Delassus, S., R. Cheynier, and S. Wain-Hobson. 1991. Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro. J. Virol. 65:225-231. 4. Domingo, E. 1989. RNA virus evolution and the control of viral disease. Prog. Drug Res. 33:93-133. 5. Domingo, E., J. J. Holland, and P. Ahlquist (ed.). 1988. RNA genetics. CRC Press, Inc., Baton Rouge, La. 6. Epstein, L. G., C. Kuiken, B. M. Blumberg, S. Hartman, L. R. Sharer, M. Clement, and J. Goudsmit. 1991. HIV-1 V3 domain variation in brain and spleen of children with AIDS: tissuespecific evolution within host-determined quasispecies. Virology 180:583-590. 7. Fujita, T., H. Shibuya, T. Ohashi, K. Yamanishi, and T. Taniguchi. 1986. Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes. Cell 46:401-407. 8. Goodenow, M., T. Huet, W. Saurin, S. Kwok, J. Sninsky, and S. Wain-Hobson. 1989. HIV-1 isolates are rapidly evolving quasispecies: evidence for viral mixtures and preferred nucleotide
substitutions. J. Acquired Immune Defic. Syndr.
2:344-352.
9. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.
VOL. 65, 1991
10. Hadzopoulou-Cladaras, M., B. K. Felber, C. Cladaras, A. Athanassopoulos, A. Tse, and G. N. Paviakis. 1989. The rev (trslart) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J. Virol. 63:1265-1274. 11. Hu, W. S., and H. M. Temin. 1990. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc. Natl. Acad. Sci. USA 87:1556-1560. 12. Kozak, M. 1989. The scanning model for translation: an update. J. Cell Biol. 108:229-241. 13. Lu, X., J. Heimer, D. Rekosh, and M. L. Hammarskjold. 1990. Ul small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proc. Natl. Acad. Sci. USA 87:7598-7602. 14. Malim, M. H., S. Bohnlein, J. Hauber, and B. R. Cullen. 1989. Functional dissection of the HIV-1 rev transactivator: derivation of a transdominant repressor of rev function. Cell 58:205214. 15. Malim, M. H., J. Hauber, S. Le, J. Maizel, and B. R. Cullen. 1989. The HIV-1 rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral RNA. Nature (London) 338:254-256. 16. Meyerhans, A., R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjo, and S. Wain-Hobson. 1989. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58:901-910. 17. Meyerhans, A., J. P. Vartanian, and S. Wain-Hobson. 1990. DNA recombination during PCR. Nucleic Acids Res. 18:16871691. 18. Meyerhans, A., and S. Wain-Hobson. Unpublished data. 19. Perkins, A., A. W. Cochrane, S. M. Ruben, and C. A. Rosen. 1989. Structural and functional characterization of the human immunodeficiency virus rev protein. J. Acquired Immune Defic. Syndr. 2:256-263. 20. Rosen, C. A., J. G. Sodroski, K. Campbell, and W. A. Haseltine. 1986. Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human
NOTES
21.
22.
23.
24.
25.
26. 27.
28. 29.
4507
T-cell lymphotropic virus type III transactivator genes. J. Virol. 57:379-384. Rosen, C. A., E. Terwillinger, A. Dayton, J. G. Sodroski, and W. A. Haseltine. 1988. Intragenic cis acting art gene responsive sequences of the human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85:2071-2075. Saag, M., B. H. Hahn, J. Gibbons, Y. Li, E. S. Parks, W. P. Parks, and G. M. Shaw. 1988. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature (London) 334: 440-444. Schwartz, S., B. K. Felber, D. M. Benko, E. M. Fenyo, and G. Pavlakis. 1990. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J. Virol. 64:2519-2529. Simmonds, P., P. Balfe, C. A. Ludlam, J. 0. Bishop, and A. J. Leigh Brown. 1990. Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1. J. Virol. 64:5840-5850. Sodroski, J., W. C. Goh, C. A. Rosen, A. Dayton, E. Terwillinger, and W. A. Haseltine. 1986. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature (London) 321:412-417. Trono, D., M. B. Feinberg, and D. Baltimore. 1989. HIV-1 gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 59:215-223. Vartanian, J.-P., A. Meyerhans, B. Asjo, and S. Wain-Hobson. 1991. Selection, recombination, and G-+A hypermutation of human immunodeficiency virus type 1 genomes. J. Virol. 65: 1779-1788. Wain-Hobson, S., P. Sonigo, 0. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17. Wain-Hobson, S., J.-P. Vartanian, M. Henry, N. Chenciner, R. Cheynier, S. Delassus, L. Pedroza Martins, M. Sala, M.-T. Nugeyere, D. Guetard, D. Klatzmann, J.-C. Gluckman, W. Rozenbaum, F. Barre-Sinoussi, and L. Montagnier. 1991. LAV revisited: origins of the early HIV-1 isolates from the Institut Pasteur. Science 252:961-969.
JOURNAL OF VIROLOGY, Aug. 1991, p. 4502-4507 0022-538X/91/084502-06$02.00/0 Copyright C 1991, American Society for Microbiology
Independent Fluctuation of Human Immunodeficiency Virus Type 1 rev and gp4l Quasispecies In Vivo LIVIA PEDROZA MARTINS,' NICOLE CHENCINER,1 BIRGITTA ASJO,2 ANDREAS MEYERHANS,"3 AND SIMON WAIN-HOBSON'*
Laboratoire de Retrovirologie Moleculaire, Institut Pasteur, 75724 Paris, France'; Department of Virology, Karolinska Institute, S-10521 Stockholm, Sweden2; and Department of Virology, Institut for Medical Microbiology and Hygiene, University of Freiburg, D-7800 Freiburg, Germany3 Received 4 March 1991/Accepted 15 May 1991
The human immunodeficiency virus type 1 (HIV-1) overlapping rev and env coding sequences have been examined from sequential peripheral blood mononuclear cell DNA samples from one individual. These were the same DNA samples from which sequence data for the tat and nef/long terminal repeat loci have been derived and span a 4-year period. The rev/env sequences were established by sequencing cloned polymerase chain reaction products. The structure of the populations of rev protein sequences increased in complexity with disease, while those of the corresponding env sequences remained complex. This suggests that the rev and env populations evolved differently, probably reflecting different selection pressures. No defective rev variants encoded substitutions in residues 76 through 79, indicating that the experimental finding of down regulation of rev activity by competitive inhibition may not necessarily occur in vivo. After having analyzed three HIV loci (15% of the genome) from the same individual over 4 years, it is clear that no two loci evolved similarly, indicating the difficulties in comparing data from different loci.
while inactivating rev function, resulted in mutants capable of competitively inhibiting the native rev protein (14). In the present study, two questions were addressed. First, since this exon overlaps completely the gp4l region of the env ORF and the second tat exon, could the amino acid sequences evolve differently? Second, could defective rev gene products (14) be used as a means of autoregulation of HIV, as has been shown experimentally? Consequently, a longitudinal structure-function study of rev quasispecies in vivo as well as an ex vivo isolate was undertaken with the same DNA samples from the Swedish patient. The clinical and immunological data associated with the blood samples (Li, L2, L3, L5, and L6) and virus isolation (V6) have been previously described (3, 16). Suffice it to say that Li, L2, L3, L5, and L6 were taken in June 1985, March 1986, June 1986, February 1989, and June 1989, respectively. V6 corresponds to the HIV-1 isolate derived from cocultivation of the L6 sample with donor peripheral blood mononuclear cells. Total DNA was extracted as previously described (3, 16). The segment of the HIV-1 genome amplified from patient DNA is shown in Fig. 1A. It was decided not to amplify the 3-kb fragment encoding the two rev coding exons for three reasons. First, natural recombination between HIV-1 genomes will probably uncouple any possible cis evolution of the two exons (3, 11). Second, it was important to avoid polymerase chain reaction (PCR)-mediated recombination (17). This was shown to be sequence specific. The probability of generating PCR-mediated recombinants would surely have been greater upon amplification of the 3-kb fragment, as opposed to the 300-bp test fragments (17). Third, the first rev coding exon quasispecies were already established as part of the preceeding study of the tat gene and already shown to be reasonably homogeneous. Thus, the second exon of the rev gene was amplified as part of a larger 411-bp fragment from patient DNA by using primers REV3 and REV5 (Fig. 1A). The DNA plus strand primer REV3 (5'-CACCTCGAGTGATAGTAGGAGGCTT
RNA viruses have been described as populations of viral quasispecies (5), waiting to be selected (4). This feature is rooted in the elevated viral polymerase nucleotide misincorporation rate. Reassortment and recombination can contribute significantly to the heterogeneity of an isolate (11). The quasispecies nature of human immunodeficiency virus (HIV) in vivo and ex vivo has been intensively examined (6, 8, 16, 22, 24). Sequence analyses have described the evolution of HIV proviral sequences in vivo, thus overcoming the selective constraints of ex vivo culture upon the quasispecies (3, 16, 27). A longitudinal study of the HIV-1 proviral DNA sequences from a Swedish patient has been undertaken. Thus far, the tat and nefllong terminal repeat (LTR) loci have been analysed for the period June 1985 to June 1989 (3, 16). Disease progression was not correlated with the appearance of more efficient tat or LTR sequences. These and other studies have revealed considerable proportions of functionally defective genomes (8, 16). Here, we have focused on the second rev coding exon of HIV-1. The rev protein is a small molecule of 116 residues encoded by two exons specifying residues 1 through 26 and 27 through 116 (25). It is essential to viral replication and is involved in directing high-molecular-weight (i.e., gaglpol and env) mRNAs to the cytoplasm either by aiding transport (15) or by modulation of splicing (1, 13). The rev protein acts via a complex RNA secondary structure called the revresponsive element located in the env open reading frame (ORF) (10, 15). At least three important functional regions of the rev gene have been identified. The first is the amino terminus, which is thought to be involved in binding to the rev-responsive element (14, 19). The second is a highly basic sequence (residues 35 through 50), which is involved in targeting the rev protein to the nucleus or nucleolus (14, 19). Substitutions in the third region (residues 76 through 79), genomes, or
*
Corresponding author. 4502
VOL. 65, 1991
NOTES
L---L.
-
Il
I LTR
vI
Da
4503
EJEJE~~.rev.SI pi0 env u I%a env I I i
r~~~~~ -"
A
PBMC DNA
-_
SA
TAG REV 5
SA
TAG
REV 3
5
HIV-1 LAI ATG
SD
REV IB
C
EXPRESSION VECTOR
SV40 E
REV2
REV 4
REV 3
Hindill
L===;
Sacl Bgill/BamHI
Xho
HUV I-Oly A I
ATG
SD
SA
TAG
FIG. 1. PCR amplification of the HIV-1 rev gene and construction of chimeric rev expression vector. The organization of the HIV-1 genome is shown at the top of the figure. (A) Segment (stippled box) amplified from fresh peripheral blood mononuclear cell DNA (Li, L3, L6) and from the V6 isolate by using primers REV3 and REV5. The amplified region encoded both second coding exons of rev and tat and a fragment of the gp4l gene. (B) The regions harboring the first and the second coding exons of rev (open boxes) were amplified from HIV-1 Lai plasmid DNA by using PCR primers REV1B, REV2, REV3, and REV4. The major rev splice donor and acceptor sequences are indicated by SD and SA, respectively. ATG and TAG indicate the rev gene start and stop codon. (C) The amplified fragments of HIV-1 Lai were cloned into the pASA7 expression vector containing the simian virus 40 early promoter and the hepatitis B virus poly(A) segment. The restriction sites indicated were those used in its construction and were introduced into the PCR primers. DNA fragments corresponding to rev variants (stippled box) were then subcloned by replacement of the corresponding HIV-1 Lai fragment.
GG) and minus strand primer REV5 (5'-CCGAGCTCTATA ACCCTATCTGTCC) mapped to positions 7880 to 7897 and 8308 to 8291, respectively, on the HIV-1 Lai sequence (28). In light of recent findings, LAV has been renamed HIV-1 Lai (29). The primers carried XhoI and SacI restriction sites (underlined), respectively. Optimized PCR conditions for these primers were 1.25 mM MgCl2, 25 pmol of each primer, and 2.5 U of Taq polymerase per 100-,I reaction. The DNA was amplified for 42 cycles. Thermal cycling parameters were as follows: denaturation, 95°C, 30 s; annealing, 50°C, 25 s; and elongation, 72°C, 2 min. Amplification was followed by a final 10-min step at 72°C. High-temperature Taq addition was employed. PCR products were cloned as usual (3). Plaques were screened by hybridization with a revspecific 32P-radiolabelled probe. Twenty M13 recombinants were picked from each sample and grown up for sequencing. Single-stranded DNA was sequenced by the dideoxy chain termination method with fluorescent M13 universal primers, 7-azadGTP, and Taq polymerase. The products were resolved by an Applied Biosystems 370A DNA sequencer. The error associated with PCR amplification is such that 1 to 1.5 amino acid substitutions per 20 sequences may be due to Taq polymerase. rev quasispecies. The populations of rev amino acid sequences derived from samples Li, L3, and L6, as well as the V6 ex vivo sample, are shown in Fig. 2. One sequence, identified by the symbol *, was common to all four quasispecies (L102R, L304R, L602R, and V624R), while two other sequences were common to Li and L3 only. That a major form persisted for 4 years is unusual. Previously, the major form was invariably derived from a minor form present in the preceding quasispecies (3, 16). The structure
of the population of Li rev sequences was most remarkable, being composed of three major (i.e., >5%) forms unaccompanied by any minor form, at least given the resolution of this study. The number of variants in the quasispecies appeared to increase from Li (3 sequences) to L3 (9 sequences) to L6 (13 sequences). The internal rev sequence variation for each of the three quasispecies was between 1 and 5%. The isolation of HIV resulted in the outgrowth of a minor form and homogenization of the quasispecies, as evidenced by comparing the L6 and V6 data. Relatively few defective rev sequences were identified in this exon. They included L603R (W45--R45) and L302R. The latter encoded a substitution in the major rev splice acceptor (i.e., AG/
AC-*AA/AC). The previous study of the tat sequences from the same patient (16, 18) naturally included sequence data for the first rev coding exon. The corresponding populations of protein sequences specified by this exon are shown in Fig. 3. A single form was dominant in all the samples. Noteworthy was the L5 quasispecies, in which a full 30% of sequences encoded a defective rev sequence due to a G->A transition in the initiator methionine codon. The rev splice acceptors and donor sequences 5' and 3' to this coding region were conserved (23). env quasispecies. The second rev coding exon is overlapped by part of the env ORF corresponding to the cytoplasmic tail of the gp4l transmembrane protein (Fig. 1). Consequently the nucleotide sequence of this region might be more constrained than any of the other regions examined to date. Analysis of the same nucleotide sequence, but this time in the gp4l ORF, yielded a very different profile. Thus the Li, L3, and L6 env quasispecies were all complex, being
4504
J. VIROL.
NOTES
Freq. Rel.Act 45 55 75 65 85 95 105 115 DPPPSPEGTR QARRNRRRRW RERQRQIRSI SGWIISTYLG RPAEPVPLQL PPLERLTLDC SEDCGTSGTQ GVGSPQILVE SPTVLESGTK
35
O L105R *
L102R
*
L103R
*
L304R L303R
................
* L319R
.......... .......... ..........
L302R
§.........
L305R L323R L327R
.......... .......... .......... .......... ..........
L602R
...
0
L31OR
L315R
*
L605R O L620R
*
L603R L607R L61OR L611R L614R L615R L619R
.... .... I.....
....I..... ....I.....
*
.....
V630R
.
T T
.......
.......
.
.
T
......
.............
.......
....I..... ....I..... ....I.....
...
N N
...........
.....
.
...........
..........
.......
..........
..........
......
...N...... ...N...... ...N...... ...N...... ...N...... ...N...... ...N...... ...N...... ...N......
A... T.. ....... .N.... H
.N...I....
* V601R O V607R V621R
.
.
.
.... I.....
.... I.....
V624R
.
N
..T..A .TA......
.
1.6 1 .3 1.6
40 1.3 20 1.6 10 1.6 5 0.1 .A 5 2.6 2.6 5 5 0.9 ... .........R 2.7 5 .......... 5 N.D. N.... ..V . .......... ...
.A...
.... I.....
. .
...........
.......... . ....N...
.... I.....
L621R L625R
V606R
N...... N .
.......... .............
(%) 45 40 15
...N ...... ...N ...... ...N ......
N...T..T .. ........ N. T. .
..T
.
.
.
N N N N N N
G..K
.
.......
......
FIG. 2. Genetic and functional complexity of rev quasispecies. The
...
sequences,
.
.
.
.
.
.
.
.
.
.
R..
.
.
.
.
A
......
......
..........
..........
..........
.
..........
.
were
5 5
0.6 0.8
25 10
..........
given here in the one-letter code,
5
1 .0 1.3 1.3 0.6
50
..........
. . .....
.N.... 0.
20 1.3 20 1.4 15 1.3 ....A. 5 0.04 ....A. 5 N.D ....... 5 3.0 5 0.9 ..... 2.0 5 5 1.7 1.0 5 1.8 5 A A A A 5 0.8 .
......
aligned with respect
to the most abundant sequence in the initial sample Li. Only the differences were scored, a dot indicating sequence identity; § indicates a G-*A transition which abolished the main splice acceptor. The four populations correspond to samples Li, L3, L6, and V6. The symbols (* 0 @0 *) on the left identify the same protein sequence in different samples. The activities of 21 of 23 rev variants were determined in a transient CAT assay. N. D., not determined. CAT activities relative to HIV-1 Lai are shown in the last column. They represent the means of at least three experiments, except for L610, L614, L615, and L621, which represent the means of two experiments. CAT activities after cotransfection of pIllIAR and HIV-1 Lai homologous plasmids were typically between 10 to 20%, whereas the negative controls were of the order of 1 to 2%. Li
made up of 16, 13, and 13 sequences, respectively, none of which exceeded 20% (Fig. 4). Only three sequences were common to Li and L3. None of the 13 distinct L6 sequences were found in the preceeding Li or L3 samples. The internal env sequence variation for each of the three quasispecies was between 1 and 5%. Finally the same locus encoded 30 amino acid residues corresponding to the second tat coding exon. Despite its small size, fluctuations in the populations of sequences were observed (data not shown). This contrasts with the striking stability and homogeneity of the data from the first rev coding exon (Fig. 3). A number of features in the data presented above might suggest that stronger selection pressures were operative on the rev than on the gp41 env locus. First, in both rev exons a major form persisted for 4 years. Second, Li rev quasispecies was much more homogeneous than the corresponding env quasispecies, and finally, the complexity of the rev
MAGRSGDSDE ELLRTVRLIK FLYQS 80%
.
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.......
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95%
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87%
0
0
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.......... ..........
.......... .......... R. 7%
L5
58% 31% .......
N.. 11% A .........
.....
L6. .......... 95% .......... .....
0
5%
.......... .........
quasispecies increased during disease progression.
This study represents the extension of analyses of the HIV-1 regulatory gene sequences within a Swedish HIV-1seropositive patient. The preceeding studies described the tat (16) and neftLTR loci (3). Figure 5 attempts to describe the evolution of HIV-1 quasispecies with time. In fact, it represents a simplification, since the notion of a quasispecies must describe not only the number and frequencies of the component sequences but also the sequence variation. Only the rev quasispecies increased in complexity during disease progression. Every locus evolved differently, and within each locus, every ORF evolved independently. In view of this and the fact that the tat, revlenv and neflLTR repre-
V6
80%
........ ..........
.......
0
N.. ... A ..........
.....
........ I. ..........
.....
5%
FIG. 3. Sequence variability of the rev amino terminus. The nucleotide sequence data for Li, L2, and L3 have already been published (16). L5 (February 1989), L6 (June 1989), and V6 (virus derived from sample L6) samples corresponded to subsequent time points (18). rev protein sequences, given here in the one-letter code, were aligned to the dominant form present in Li. Dots indicates sequence identity. The frequency of each sequence is given to the right of the figure. Symbols (- A *) indicate identical protein sequences.
.
VOL. 65, 1991
..
NOTES
4505
815 746 806 736 77 6 726 796 786 7 56 76 6 YSPLSFQTRL PAPRGPDRPD GTEEEGGERD RIDRSGRLVDG LLAL IWDDLR SLCLFSYHRL RDLLSIAARI VELLGRRGWE VLKYWWNLLQ YWSQELKNR I..... L106E . ........
L105E
L108E L11OE L107E L102E L103E L104E L109E L112E L114E LllSE L117E L118E L121E L137E
L310E L321E L308E L323E L311E L302E L303E L304E L305E L314E L315E L319E L324E
L602E L605E L603E L608E L610E L611E L614E L615E L619E L620E L621E L624E L625E
.
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5% 5%
FIG. 4. Sequence complexity of env quasispecies in vivo. The sequences, given here in the one-letter code, were aligned with respect to the most abundant protein sequence in the initial sample, Li. Only the differences were scored, a dot indicating sequence identity. The three populations correspond to samples Li, L3, and L6. The frequency of a given protein sequence is shown on the right. The symbols (*- V) on the right identify the same protein sequence in different samples.
sented together only 15% of the HIV genome, we feel that it is not prudent to extrapolate from these data to other regions of the genome. rev function. Several studies have shown that certain segments of the HIV-1 genome harbor up to 15% of defective genomes (8, 16). Clearly, as evidenced by the high frequency of rev mutant sequences in the L5 sample, the proportion can be even greater. The contribution of defective genomes to the overall replication of HIV-1 has been addressed only in the context of particular experimental systems (14, 26). In order to test the activity of the rev variants, an expression vector was first constructed from the two coding exons of HIV-1 Lai. The backbone of the vector was derived from pASA7 (2), including the simian virus 40 early promoter and the hepatitis B virus poly(A) sequences. The first rev coding exon was amplified from the HIV-1 Lai clone by using the REVlB and REV2 pair of primers (Fig. 1B). Their sequences and coordinates were: REV1B, 5'-CCGAAGCTIT GAAATGGCTGGAAGAAGCG, positions 5548 to 5566, and REV2, 5'-CACCTCGAGACCACACAACTATTGC, positions 5710 to 5694. Furthermore, the primers encoded HindIII and XhoI restriction sites, respectively (underlined). In order to ensure efficient translation of the transcribed rev
the nonoptimal Kozak sequence (12), surrounding the natural initiator methionine codon (ATG), was changed from CCTATGG to GAAATGG by incorporation of this latter sequence into the REVlB primer (shown in boldface). The vpulenv splice acceptor sequence mapped within the REVlB primer sequence (23). To eliminate this sequence, the allimportant AG dinucleotide was substituted in the primer by a TG (also in boldface), without changing the rev coding sequence. The second coding exon of HIV-1 Lai was amplified by using the REV3 and REV4 primer pair (Fig. 1B). The sequence of REV4 was 5'-CCAGATCTGAGCTCTATAAC CCTATCTGTC, and it mapped to 8306 to 8291. It included both BglII (underlined) and Sacl (doubly underlined) restriction sites at its 5' end. Approximately 100 ng of HIV-1 Lai plasmid DNA was amplified for 15 cycles. The two amplified fragments were cloned into the HindlIl and BamHI sites of pASA7 (Fig. 1C). Eighty bases 3' to the rev splice donor and 93 bases 5' to the splice acceptor were included, so as to ensure correct splicing. This construct was designated pASArev. DNA fragments from 21 of the rev variants were subcloned into the pASArev vector. These subclones therefore represented chimeras of the first coding exon of HIV-1 Lai rev and the second rev exon from the Swedish patient
gene,
J. VIROL.
NOTES
4506
201 nelI
(206 aa)
41(99 aa) U)
v2(91 aa)
ut
r.
10-i 2(30 aa)
0
7
6 z
t1(72 aa)
Li
0
L2 L3 10
20
30
rl(25 aa) L5 L6 50 40
Time/months
FIG. 5. Complexity of different segments of t the HIV genome during disease progression. The number of dis tinct protein sequences per locus analyzed (i.e., tat, revlenv o r neflLTR) as a function of time (months) is given. Thus, rev 1 and tat 1 are deduced from the same locus (16, 18), as are rev 2, tat 2 and gp41 (this study), and nef (3). Because the lengths of the protei tn sequences are different, the number of residues is indicated i n parentheses to facilitate comparisons.
(Fig. 1C). The subclones were verified by sequencing the plasmid DNA. Rev activity was determined using pIlAlR, a rev-dependent chloramphenicol acetyltransferase (C)AT) expression deTreviouslsde plasmid (21) and Jurkat-tat T cells (20), as previously scribed. For each assay, approximately 4 x 106 cells were cotransfected with 3 ,ug of pIIIAR and 10 ,ug of recombinant rev plasmid by using the DEAE-dextran r nethod (7). All experiments included pASArev as a positive control and two negative controls: pSV2gpt and a derivatiNve of pASArev which carried a + 1 frameshift mutation in the first rev coding exon. Cells were harvested 48 h posttransfecction and lysed. CAT assays were conducted as previously described (9) after having normalized lysates to equal prc tein concentration (by using the Bio-Rad protein assay). The activities of the heterologous rev prod [ucts, relative to that of the homologous HIV-1 Lai construc St, are given on the right of Fig. 2. The values given were tthe means of at least two independent transfections, and the y represent the percentage of acetylation given by a rev vairiant relative to that of HIV-1 Lai, i.e., plasmid pASArev. [he activities of all the major forms were comparable to that c f the pASArev. Site were Variants lacking the serine (S99) phosphory ,lation found in all samples (Fig. 2), and their a(ctivity was not affected, confirming previous findings that rphosphorylation is not essential to rev activity (14). Of the n uinor forms, the activity of L603R (W45-*R45) was in th e range of the negative control, while that of the L302R variant, which lacked the splice acceptor sequence, was gireatly impaired. Site-directed mutagenesis of the third domzain (residues 76 through 79) resulted in defective rev prote ins which were able, nonetheless, to function as trans-domin iant inhibitors of the native rev protein (14, 19). In the present study, only one variant, V606R, encoded a substitution (R8 0-i>G80) in this domain which, however, did not alter rev activity significantly. The defective rev genes identified he]re (Fig. 2 and 3)
AtionhsitewSrev
antudy,inhii
ofe
encoded substitutions in the other domains. This would indicate that down regulation of rev activity by competitive inhibition has not been seized upon by HIV-1 in vivo as a possible mode of autoregulation. All the major forms tested, whether the gene were rev or tat or the sequences were from the LTR, had activities comparable to that of the corresponding HIV-1 Lai gene or sequence. However, it is possible that our experimental approaches, i.e., transfection of mammalian cells and transient expression of reporter genes such as CAT or secreted alkaline phosphatase, may not be sufficiently sensitive to detect subtle differences in gene function. This could be overcome by cloning the variants back into an infectious molecular clone. Any subtle fitness advantage of a particular variant with respect to its parent might be evident only after multiple rounds of replication. Here a problem arises. Which infectious molecular clone should be used? Should it be one of the molecular clones derived from virus adapted to established cell lines? When taken together, the three studies of HIV-1 regulatory genes and sequences derived from the patient suggest that despite the disease stage, isolation of HIV-1 always resulted in the outgrowth of a minor form in vivo. Perhaps
the single most important problem to be solved is the development of culture techniques which deform as little as possible the HIV-1 quasispecies. Until this is achieved, the quasispecies nature of HIV-1 will almost certainly constitute an important barrier to a detailed molecular definition of HIV-associated pathogenesis.
We thank Rdmi Cheynier and John Sninsky for help and discussions and Craig Rosen for the pIIIAR plasmid. L.P.M. was supported by a fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. This work was supported by grants from the Institut Pasteur, de Recherches sur le SIDA, and the Swedish l'Agence Nationale Medical Research Council.
REFERENCES 1. Chang, D. D., and P. A. Sharp. 1989. Regulation of HIV rev depends upon recognition of splice sites. Cell 59:789-795. 2. Chenciner, N., F. Delpeyroux, N. Israel, M. Lambert, A. Lim, R. Streeck, and J. F. Houssais. 1990. Enhancement of gene expression by somatic hybridization with primary cells: high-level in monkey Vero cells B surface antigen hepatitishepatocytes. of theprimary synthesis Bio/Technology 8:858-862. by fusion with 3. Delassus, S., R. Cheynier, and S. Wain-Hobson. 1991. Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro. J. Virol. 65:225-231. 4. Domingo, E. 1989. RNA virus evolution and the control of viral disease. Prog. Drug Res. 33:93-133. 5. Domingo, E., J. J. Holland, and P. Ahlquist (ed.). 1988. RNA genetics. CRC Press, Inc., Baton Rouge, La. 6. Epstein, L. G., C. Kuiken, B. M. Blumberg, S. Hartman, L. R. Sharer, M. Clement, and J. Goudsmit. 1991. HIV-1 V3 domain variation in brain and spleen of children with AIDS: tissuespecific evolution within host-determined quasispecies. Virology 180:583-590. 7. Fujita, T., H. Shibuya, T. Ohashi, K. Yamanishi, and T. Taniguchi. 1986. Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes. Cell 46:401-407. 8. Goodenow, M., T. Huet, W. Saurin, S. Kwok, J. Sninsky, and S. Wain-Hobson. 1989. HIV-1 isolates are rapidly evolving quasispecies: evidence for viral mixtures and preferred nucleotide
substitutions. J. Acquired Immune Defic. Syndr.
2:344-352.
9. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.
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10. Hadzopoulou-Cladaras, M., B. K. Felber, C. Cladaras, A. Athanassopoulos, A. Tse, and G. N. Paviakis. 1989. The rev (trslart) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J. Virol. 63:1265-1274. 11. Hu, W. S., and H. M. Temin. 1990. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc. Natl. Acad. Sci. USA 87:1556-1560. 12. Kozak, M. 1989. The scanning model for translation: an update. J. Cell Biol. 108:229-241. 13. Lu, X., J. Heimer, D. Rekosh, and M. L. Hammarskjold. 1990. Ul small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proc. Natl. Acad. Sci. USA 87:7598-7602. 14. Malim, M. H., S. Bohnlein, J. Hauber, and B. R. Cullen. 1989. Functional dissection of the HIV-1 rev transactivator: derivation of a transdominant repressor of rev function. Cell 58:205214. 15. Malim, M. H., J. Hauber, S. Le, J. Maizel, and B. R. Cullen. 1989. The HIV-1 rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral RNA. Nature (London) 338:254-256. 16. Meyerhans, A., R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjo, and S. Wain-Hobson. 1989. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58:901-910. 17. Meyerhans, A., J. P. Vartanian, and S. Wain-Hobson. 1990. DNA recombination during PCR. Nucleic Acids Res. 18:16871691. 18. Meyerhans, A., and S. Wain-Hobson. Unpublished data. 19. Perkins, A., A. W. Cochrane, S. M. Ruben, and C. A. Rosen. 1989. Structural and functional characterization of the human immunodeficiency virus rev protein. J. Acquired Immune Defic. Syndr. 2:256-263. 20. Rosen, C. A., J. G. Sodroski, K. Campbell, and W. A. Haseltine. 1986. Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human
NOTES
21.
22.
23.
24.
25.
26. 27.
28. 29.
4507
T-cell lymphotropic virus type III transactivator genes. J. Virol. 57:379-384. Rosen, C. A., E. Terwillinger, A. Dayton, J. G. Sodroski, and W. A. Haseltine. 1988. Intragenic cis acting art gene responsive sequences of the human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85:2071-2075. Saag, M., B. H. Hahn, J. Gibbons, Y. Li, E. S. Parks, W. P. Parks, and G. M. Shaw. 1988. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature (London) 334: 440-444. Schwartz, S., B. K. Felber, D. M. Benko, E. M. Fenyo, and G. Pavlakis. 1990. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J. Virol. 64:2519-2529. Simmonds, P., P. Balfe, C. A. Ludlam, J. 0. Bishop, and A. J. Leigh Brown. 1990. Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1. J. Virol. 64:5840-5850. Sodroski, J., W. C. Goh, C. A. Rosen, A. Dayton, E. Terwillinger, and W. A. Haseltine. 1986. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature (London) 321:412-417. Trono, D., M. B. Feinberg, and D. Baltimore. 1989. HIV-1 gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 59:215-223. Vartanian, J.-P., A. Meyerhans, B. Asjo, and S. Wain-Hobson. 1991. Selection, recombination, and G-+A hypermutation of human immunodeficiency virus type 1 genomes. J. Virol. 65: 1779-1788. Wain-Hobson, S., P. Sonigo, 0. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17. Wain-Hobson, S., J.-P. Vartanian, M. Henry, N. Chenciner, R. Cheynier, S. Delassus, L. Pedroza Martins, M. Sala, M.-T. Nugeyere, D. Guetard, D. Klatzmann, J.-C. Gluckman, W. Rozenbaum, F. Barre-Sinoussi, and L. Montagnier. 1991. LAV revisited: origins of the early HIV-1 isolates from the Institut Pasteur. Science 252:961-969.
