VIROLOGY 222, 391–400 (1996) ARTICLE NO. 0436

The Mutation Rate of Human Immunodeficiency Virus Type 1 1 View metadata, citation and similar papers at core.ac.ukIs Influenced by the vpr Gene brought to you by CORE provided by Elsevier - Publisher Connector LOUIS M. MANSKY2

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, Wisconsin 53706 Received March 19, 1996; accepted June 14, 1996

A system has been designed to study the in vivo forward rate of mutation of human immunodeficiency virus type 1 (HIV- 1) during one round of replication. A HIV-1 shuttle vector was used that contained the lacZa peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 1 1005 mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 with sense-strand RNA and DNA templates of the lacZa peptide gene in a cell-free system. To test the hypothesis that the vpr gene product might, at least in part, account for the lower mutation rate observed in vivo, a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-type vpr gene product. A vpr0 shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of the vpr0 vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence of vpr on the mutation rate. Replication of a vpr0 shuttle vector in the presence of a wild-type vpr expression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that the vpr mutation could be complemented in trans. Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence of vpr on the mutation rate. ᭧ 1996 Academic Press, Inc.

INTRODUCTION the average lifetime of an HIV-1-infected cell is less than 1 to 2 days (Ho et al., 1995; Wei et al., 1995). The Retroviridae family of RNA viruses replicate A system has been developed to measure the forward through a DNA intermediate (Baltimore, 1970; Temin and mutation rate of HIV-1 with a vector containing the lacZa Mitzutani, 1970). The viral RNA is copied into DNA by the peptide gene as a reporter for mutations (Mansky and viral-encoded enzyme reverse transcriptase in a process Temin, 1995). This system allows for the study of muta- known as reverse transcription. The process of reverse tions that occur during a single round of HIV-1 replication. transcription is known to be error-prone, contributing to The mutation rate of HIV-1 in this system was determined the high genetic variability of (Coffin, 1992; to be 3 1 1005 mutations per target base pair per cycle Holland et al., 1992; Katz and Skalka, 1990; Temin, 1961). in HeLa cells (Mansky and Temin, 1995) and 4 1 1005 The error-prone nature of reverse transcription has been mutations per target base pair per cycle in a T-lymphoid proposed to be due to abnormal strand transfers that cell line (Mansky, 1996). The most commonly detected can occur during this process (Temin, 1993). The level mutations were base substitution mutations (G to A and of genetic variation of human immunodeficiency virus C to T transition mutations) and frameshift mutations (01 type 1 (HIV-1) is high (Alizon et al., 1986; Balfe et al., frameshifts in runs of Ts and As). This mutation rate is 1990; Cheng-Mayer et al., 1991; Hahn et al., 1986; Levy, maximally 5% of the reported error rates for purified HIV- 1993; Starcich et al., 1986; Wong-Staal, 1990) relative to 1 reverse transcriptase with a sense-strand RNA and some retroviruses in other genera (Dube et al., 1993; DNA template in cell-free studies, indicating that the mu- Ratner et al., 1991; Willems et al., 1993). Genetic variation tation rate of HIV-1 is less than that predicted by the of retroviruses can be viewed as the composite of several measured fidelity of purified HIV-1 reverse transcriptase variables: (1) the mutation rate per replication cycle, (2) (Boyer et al., 1992). the number of replication cycles, (3) the fixation rate of The difference between the in vivo mutation rate of mutations, and (4) the rate of recombination (Coffin, 1990; HIV-1 and the error rate of purified HIV-1 reverse tran- Hu and Temin, 1990; Zhang and Temin, 1993). It has scriptase may be due to several possibilities. One hy- been argued that HIV-1 completes about 300 or more pothesis is that, in vivo, protein factors and reverse tran- cycles per year (Coffin, 1995), based on the reports that scriptase together contribute to the accuracy of reverse transcription; in the absence of these factors, the error 1 This paper is dedicated to the memory of Howard M. Temin. rate of purified reverse transcriptase is higher. Protein 2 Fax: (608) 262-2824; E-mail: [email protected]. factors that are viral proteins would be predicted, like

0042-6822/96 $18.00 391 Copyright ᭧ 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. HIV-1 shuttle vector used for mutation rate studies. The HIV-1 shuttle vector is shown in the proviral DNA form. Solid black boxes represent the HIV-1 long terminal repeats (LTRs). Solid black lines indicate viral sequences. Rectangular boxes above the solid black lines indicate viral coding sequences, with the relative locations of the boxes corresponding to the translational reading frame. Retroviral genes are indicated as vif, vpr, , , vpu, and . Bent lines in between the coding regions for tat and rev show reading frames joined by splicing events. E indicates the location of the encapsidation signal. The simian virus 40 promoter (SV) is represented as a cross-hatched box, the neo gene is represented as a hatched rectangular box, the pACYC origin of replication is represented as the light gray box, and the lacZa peptide gene is represented as an open rectangular box with a black band representing the lac operator sequence. Dashed lines indicate deleted viral coding sequence removed by digestion with SwaI. The StuI and XhoI sites were used in the purification of the vector proviral DNA containing the neo gene, the pACYC origin of replication, and the lacZa peptide gene. reverse transcriptase, to be present in virus particles. To made by a primary/combinatorial two-step polymerase test the hypothesis that viral proteins contribute to the chain reaction (PCR) protocol (Ito et al., 1991). Oligonu- accuracy of reverse transcription, the HIV-1 vpr gene was cleotide primers that changed the start codon of the vpr mutated in order to determine the influence of vpr on the gene from ATG to ATT, but did not affect the overlapping rate of mutation. The vpr gene encodes a 96-amino-acid, vif open reading frame, were used as internal primers. nonstructural protein that is associated with virus parti- The PCR product from this reaction, containing the vpr cles (Cohen et al., 1990) and that requires the p6 region gene with the mutation of the start codon, was cloned of the gag gene for incorporation (Kondo et al., 1995; Lu into the pCRII cloning vector (Invitrogen Corp., San Diego, et al., 1995, 1993; Paxton et al., 1993). The incorporation CA). The cloned DNA was sequenced to verify the intro- of Vpr into virus particles implies a role either early in duced mutations using a nonradioactive DNA sequenc- the HIV-1 replication cycle (prior to the synthesis of new ing kit (Silver Sequencing kit; Promega Corp., Madison, viral proteins) or late in the replication cycle (during as- WI) and digested with PflMI and SalI, and the fragment sembly and budding). Specific roles of vpr include the containing the vpr gene was cloned into the PflMI and nuclear localization of the preintegration complex and SalI sites of pHIV shuttle 3.12. prevention of cell proliferation during chronic infection The construction of the Vpr virion-incorporation mutant (He et al., 1995; Heinzinger et al., 1994; Jowett et al., was made using a similar PCR mutagenesis protocol 1995; Planelles et al., 1995; Rogel et al., 1995). This study used for pHIV shuttle 3.12 vprATG0 (as described above). 0 has found that the mutation rate of a vpr vector mutant Nucleotide changes in the vpr gene of HIV shuttle 3.12 / was as much as fourfold higher than that of the vpr were made that led to changes in the coding for amino parental vector. This observation supports an early role acid 30 (alanine to phenylalanine; Vpr mutant A30F); this of vpr in the HIV-1 replication cycle. These data support residue is near the amino-terminal end of Vpr in the the conclusion that the vpr gene partially accounts for predicted a-helical region of the protein (residues 17– the lower than predicted in vivo mutation rate of HIV-1. 34). Mutagenesis of this domain has been found to affect Vpr stability and the efficiency of Vpr incorporation into MATERIALS AND METHODS virions (Mahalingam et al., 1995a,b,c). This particular amino acid substitution mutation in the vpr gene has HIV-1 vectors and expression plasmids been previously reported to limit the incorporation of Vpr The plasmid pHIV shuttle 3.12 (Fig. 1) was constructed into virus particles (Yao et al., 1995). as described (Mansky and Temin, 1995). A vpr0 derivative The HIV-1 gag– expression plasmid used, pSVgag- of pHIV shuttle 3.12, pHIV shuttle 3.12 vpr ATG0, was pol-rre-r (Smith et al., 1990), was kindly provided by David

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Rekosh, University of Virginia. The amphotropic murine mids and the HIV-1 tat expression plasmid (Step 2 cells); leukemia virus expression plasmid used, pSV-A- in complementation experiments, pCMVvpr was tran- MLV-env (Landau and Littman, 1992), was provided by siently transfected along with these plasmids. Step 2 Dan Littman, University of California, San Francisco. The clones were tested by Southern analysis to ensure that vpr expression plasmid, pCMVvpr, was constructed by only a single vector proviral DNA was present. The lacZa amplifying the vpr gene from HIV shuttle 3.12 by PCR peptide gene in the vector proviral DNA of Step 2 clones and inserting it into a eukaryotic expression vector that was sequenced to confirm that no mutations were intro- contains the CMV promotor (pCR3; Invitrogen). duced. In Step 3, vector virus was transferred to fresh HeLa target cells by cocultivation 24–48 hr after transient Cells and media transfection of helper plasmids; cells were then placed The HeLa and COS-1 cell lines used were obtained under G418 selection (Step 3 cells). Cocultivation was from the American Type Culture Collection (Rockville, used to produce Step 3 cells to maximize the number of MD) and were maintained in Temin’s modified Eagle’s Step 3 cells for analysis of the mutation rate. medium (Temin, 1968) containing 10% calf serum or 10% fetal bovine serum, respectively. Recovery of shuttle vector proviral DNA and DNA sequencing of the lacZa peptide region Transfections, infections, and cocultivations Purified genomic DNA (Sambrook et al., 1989) from HIV-1 vectors and expression plasmids were trans- pools of Step 3 clones was digested with the restriction fected into COS-1 or HeLa cells by use of DMSO/poly- enzymes StuI and XhoI, to release the neo, pACYC origin brene (Kawai and Nishizawa, 1984). HeLa cells were of replication, and lacZa peptide gene sequences from infected in the presence of polybrene (Mansky, 1994). the HIV-1 shuttle vector proviral DNA (Fig. 1). Proviral Infection of HeLa target cells was done by cocultivation DNA was purified with the Lac repressor protein (Pro- of virus-producing cells with target cells (Mansky et al., mega) as previously described (Mansky and Temin, 1995; Mansky and Temin, 1994). Briefly, virus-producing 1994). The purified proviral DNA was treated with the cells (typically 2.5 1 105 cells per 60-mm petri dish, 5 1 Klenow fragment of DNA polymerase to fill in single- 105 cells per 100-mm petri dish, or 7.5 1 105 cells per stranded ends, and was ligated and used to electropor- 150-mm petri dish) were treated with mitomycin C (10 ate competent Escherichia coli XLI Blue cells. Kanamy- mg/ml), an inhibitor of host cell DNA synthesis, for 2 hr cin-resistant bacterial colonies were selected in the at 37Њ. The cells were then washed three times with fresh presence of the 5-bromo-4-chloro-3-indolyl-b-D-thioga- medium, and HeLa target cells equivalent to the number lactopyranoside inducer. The ratio of white plus light-blue of treated virus-producing cells were added. Two days bacterial colonies to total bacterial colonies observed after cocultivation, selective medium containing G418 provided a forward mutation rate for a single retroviral was added. Control experiments were done with each replication cycle. Plasmid DNA was purified (Sambrook cocultivation experiment to ensure that mitomycin C- et al., 1989) and sequenced in the lacZa peptide gene treated, virus-producing cells did not proliferate and no region with a nonradioactive DNA sequencing kit (Pro- longer adhered to the surfaces of culture dishes. Cells mega Corp.) and by an automated DNA sequencer (Ap- expressing the neo gene were selected with the neomy- plied Biosystems). cin phosphotransferase analog, G418, until the formation of colonies (typically about 3 weeks). Cell radiolabeling and immunoprecipitation analysis Step 2 cells with HIV shuttle 3.12 or derivatives with Strategy for generating a single cycle of vpr mutations were metabolically labeled with [35S]- replication methionine (100 mCi/ml) for approximately 15 hr after The experimental protocol developed to assay a single transfection with helper plasmids. Virus particles pro- cycle of HIV-1 shuttle vector replication is shown in Fig. duced during the labeling were pelleted through a 20% 2. The protocol contains three steps. In Step 1, the HIV- sucrose cushion in a Beckman SW28 rotor for 90 min 1 shuttle vector was introduced into COS-1 cells by trans- at 4Њ. Cells and pelleted virions were lysed in immuno- fection and placed under G418 selection. Cell clones precipitation buffer (1% Triton X-100, 0.5% deoxycho- were then transiently transfected with the helper plas- late, 0.1% sodium dodecyl sulfate, and 0.2 mM phenyl- mids pSVgagpol-rre-r and pSV-A-MLV-env along with a methylsulfonyl fluoride in phosphate-buffered saline) HIV-1 tat expression plasmid, pSVtat (Kao et al., 1987; and then immunoprecipitated with a rabbit anti-Vpr se- Peterlin et al., 1986) (to aid in expression of the accessory rum (deposited by L. Ratner in the AIDS Research and genes from the shuttle vector). In Step 2, vector virus Reference Reagent Program, National Institute of Al- was harvested 48 hr posttransfection from Step 1 cells lergy and Infectious Diseases, Bethesda, MD) ab- and used to infect fresh HeLa cells. G418-resistant cell sorbed to protein A–agarose beads (Bethesda Re- clones were transiently transfected with the helper plas- search Laboratories, Gaithersburg, MD).

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FIG. 2. Experimental protocol for studying one round of HIV-1 shuttle vector virus replication. (A) Experimental protocol. In Step 1, COS-1 cells were transfected with a pHIV shuttle 3.12 or a derivative and were placed under G418 selection. These cells were transiently transfected with the HIV-1 gag–pol expression plasmid, pSVGAGPOL-rre-r, the amphotropic env gene, pSV-A-MLV-env, and a HIV-1 tat expression plasmid, pSVtat (to aid in expression of the accessory genes from the shuttle vector). Fourty-eight hours posttransfection, virus was harvested and used to infect HeLa cells. G418-resistant cell clones were transiently transfected with pSVGAGPOL-rre-r, pSV-A-MLV-env, and the tat expression plasmid (Step 2 cells). Twenty-four to 48 hr posttransfection, Step 2 cells were treated with mitomycin C and cocultivated with fresh HeLa target cells, and all cells were placed under G418 selection (Step 3 cells). (B) One round of HIV-1 shuttle vector virus replication. The steps going from a parental shuttle vector provirus in the Step 2 cell to a vector provirus in the Step 3 cell constitute a single cycle of replication. These steps include transcription of the proviral DNA by the cellular transcription machinery, packaging of the viral RNA, release of viral particles, infection of target cells, reverse transcription, and integration of newly synthesized viral DNA to generate a vector provirus.

RESULTS or 7.5 1 105 cells per 150-mm petri dish; mitomycin C is an inhibitor of host cell DNA synthesis and led to Replication of the HIV-1 shuttle vectors containing cell death of Step 2 cells as described under Materials mutations of the vpr gene and Methods) with fresh HeLa target cells to produce The HIV-1 shuttle vectors used in these studies were Step 3 cells led to titers of 8 1 102 –3 1 103 CFU/2.5 1 based on HIV shuttle vector 3.12 (Fig. 1), which contains 105 HeLa target cells. The steps going from a parental a deletion in the gag–pol and env genes with an inser- shuttle vector provirus in the Step 2 cells to a vector tion, in the env gene, of a cassette containing the neo provirus in the Step 3 cells constitute a single cycle gene, the pACYC origin of replication, and the lacZa of replication (Fig. 2). These steps include transcrip- peptide gene. These vectors replicate in mammalian tion of the proviral DNA by the cellular transcription cells as a virus and can be selected with the neomycin machinery, packaging of the viral RNA, release of viral analog G418. The vectors can replicate in E. coli as a particles, infection of target cells, reverse transcrip- plasmid and are selected using the drug kanamycin. To tion, and integration of newly synthesized viral DNA be packaged into a virus particle, these vectors are com- to generate a vector provirus. Southern analysis of plemented in trans by transient transfection of cells with total DNA from each Step 2 cell clone (after transient an HIV-1 gag–pol expression plasmid and an ampho- transfection with the helper plasmids) of HIV shuttle tropic murine leukemia virus env expression plasmid. 3.12 or derivatives was done to ensure that each cell Vector virus produced from either COS-1 or HeLa clone used contained only one provirus copy (data not cells was used to infect fresh HeLa target cells (Fig. shown). Proviral DNA from at least 5 1 105 cells of 2). Cocultivation was used to produce Step 3 cells each Step 2 cell clone was purified using the Lac because it was desirable to obtain the largest number repressor protein and introduced into E. coli to screen of Step 3 cells for analysis of the mutation rate. Cocul- for mutations in the lacZa gene region; blue colonies tivation of mitomycin C-treated Step 2 cells of HIV were observed for each Step 2 clone (data not shown), shuttle 3.12 or derivatives (typically 2.5 1 105 cells per indicating that the bacterial colonies containing vector 60-mm petri dish, 5 1 105 cells per 100-mm petri dish, proviral DNA had the wild-type lacZa gene phenotype.

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TABLE 1 screened for vpr0 shuttle vector proviral DNAs containing Mutation Frequency in Recovered Proviruses for a vpr0 mutations in the lacZa peptide gene (Table 1). Fifty of HIV Shuttle Vector 3.12 Mutant these colonies had a white or light-blue colony color phenotype. The mutation frequency was 50/3254 or 15 No. of mutants/ 1 1003 mutations per cycle. This mutation frequency was Step 2 total No. of Mutation about four times that of the mutation frequency of the Mutant clone No. bacterial colonies frequency vpr/ parental vector (24/6462 or 4 1 1003 mutations per HIV shuttle 3.12 cycle) in parallel experiments (Table 1). The mutation vpr ATG0 1 10/795 13 1 1003 frequencies of the vpr0 mutant and the parental shuttle 2 17/1025 17 1 1003 vector were significantly different (x2 Å 38; P õ 0.01). 03 3 12/881 14 1 10 To determine the types of mutations in the lacZa pep- 4 11/553 20 1 1003 Total 50/3254 15 1 1003 a tide gene and to calculate the mutations per base pair Parental, vpr/ per cycle, plasmid DNA from these clones was analyzed HIV shuttle by DNA sequencing in this region. Forty-two of the 50 vector 3.12 1 8/1383 6 1 1003 mutants sequenced from the vpr0 shuttle vector were 03 2 13/3724 4 1 10 found to contain a single mutation in the lacZa peptide 3 3/1355 2 1 1003 Total 24/6462 4 1 1003 b gene region (Fig. 3). Similar kinds of mutations were observed with the vpr/ parental vector (Fig. 3). a Average mutation frequency with a standard deviation of 3 1 1003 Eight of the 50 mutants sequenced from the vpr0 shut- mutants per cycle. tle vector had multiple mutations in the lacZa peptide b 03 Average mutation frequency with a standard deviation of 2 1 10 gene region. Most of the mutants contained multiple G mutants per cycle. to A transition mutations. These mutants are likely to have resulted from proviral DNA synthesis catalyzed by The lacZa gene region in the vector proviral DNA of an error-prone reverse transcriptase. Since the forward 05 each Step 2 clone was sequenced to confirm that no mutation rate of HIV-1 is 3 1 10 mutations per base mutations had been introduced into the lacZa gene pair per cycle (Mansky and Temin, 1995), the observation region in the steps leading to the vector provirus in of 2 or more mutations in the 280-base lacZa peptide the Step 2 cell. The templates used for DNA sequenc- gene region is indicative of hypermutation (Pathak and ing were from proviral DNA purified from Step 2 cells Temin, 1990a). using the Lac repressor protein and proviral DNAs from at least three blue colonies (from the above phe- Effect of a mutation that limits incorporation of Vpr notypic analysis in E. coli) for each Step 2 clone used; into virus particles on the mutation rate DNA sequencing indicated that each Step 2 clone con- tained the wild-type lacZa gene sequence (data not A mutation in the vpr gene was made that would shown). The vpr gene in the vector proviral DNA of the limit incorporation of Vpr into virus particles, without parental Step 2 clones was sequenced to confirm that resulting in large structural changes to the protein. Spe- no mutations had been introduced in the steps leading cifically, a vpr mutant based on HIV shuttle 3.12 was to the vector provirus in the Step 2 cell (data not made that would express Vpr with a single amino acid shown). substitution at residue 30 (changing alanine to phenyl- alanine) in the a-helical domain located near the amino Mutation frequency, type, and location observed with terminus of the protein; this mutant has been reported a vpr0HIV-1 shuttle vector to limit the incorporation of Vpr into virus particles (Yao et al., 1995). Vector virus produced from COS-1 cells A HIV-1 shuttle vector containing a mutation of the vpr was used to infect fresh HeLa target cells. Step 2 cells gene start codon was used (i.e., HIV shuttle 3.12 vpr containing this shuttle vector were transfected with the ATG0). This vector was tested in parallel with the parental helper plasmids, and were cocultivated with fresh HeLa vector (Table 1). The proviral DNA from pooled Step 3 target cells to produce Step 3 cells in parallel with cells representing over 70,000 different cell clones of the cocultivation of fresh HeLa cells with the vpr/ parental vpr0 mutant was purified with the Lac repressor protein shuttle vector. and introduced into E. coli to screen for mutations in the The proviral DNA from pooled Step 3 cells represent- lacZa gene region. Vector DNAs with mutations at target ing over 50,000 different cell clones was purified with nucleotides, previously determined to lead to a pheno- the Lac repressor protein and introduced into E. coli to typic change (Bebenek et al., 1989; Pathak and Temin, screen for mutations in the lacZa gene region. Three 1990b), were detectable as bacterial colonies with a thousand ninety-two bacterial colonies were screened white or light-blue colony color phenotype. Three thou- for shuttle vector proviral DNAs containing mutations in sand two hundred fifty-four bacterial colonies were the lacZa peptide gene (Table 2). Fourty-four of these

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FIG. 3. Plus-strand nucleotide sequence of the lacZa gene region in a HIV shuttle 3.12 derivative containing a mutation of the vpr gene start codon. The start for nucleotide numbering is the beginning of the 5؅ long terminal repeat of HIV shuttle 3.12. The start and stop codons of the lacZa open reading frame (small boxed sequences) and the lac operator sequence (large boxed sequence) are shown. Nucleotide positions of base pair substitutions (letters above the sequence), /1 frameshifts (letters with ᭢ above the sequence), 01 frameshifts (᭞ above the sequence), and deletions (solid black lines with arrows above or below the sequence and adjacent to the deletion names) are indicated. Mutations from the same mutant are designated with a Greek letter adjacent to each mutation; identical Greek letters indicate mutations from the same mutant. The locations of base-substitution, frameshift, and deletion mutations in the parental vector, HIV shuttle vector 3.12, from parallel experiments (see Table 1) are indicated below the nucleotide sequence. colonies had a white or light-blue colony color pheno- These cells were then cocultivated with fresh HeLa target type. The mutation frequency was 44/3092 or 14 1 1003 cells to produce Step 3 cells along with parallel cocultiva- mutations per cycle. The mutation frequency of the vpr/ tion experiments using the vpr/ parental vector. parental vector in parallel experiments was 10/2450 or The proviral DNA from pooled Step 3 cells representing 4 1 1003 mutations per cycle. The mutation frequency of over 50,000 different cell clones was purified with the Lac the shuttle vector containing the amino acid substitution repressor protein and introduced into E. coli to screen for in Vpr was significantly different from that obtained with mutations in the lacZa gene region. Two thousand nine the parental vector (x2 Å 14; P õ 0.01), but was not hundred ninety-two bacterial colonies were screened for different than the mutation frequency of the vpr0 vector vpr-complemented shuttle vector proviral DNAs containing (x2 Å 0.14; P ú 0.1). These results indicate the require- mutations in the lacZa peptide gene (Table 3). Nineteen ment of efficient Vpr incorporation in order to observe of these colonies had a white or light-blue colony color the influence of the vpr gene on the rate of mutation. phenotype. The mutation frequency for the vpr-comple- mented shuttle vector was 19/2992 or 6 1 1003 mutations / Complementation in trans of mutations in the vpr per cycle. The mutation frequency of the vpr parental vec- 03 gene tor done in parallel experiments was 11/2074 or 5 1 10 mutations per cycle. The mutation frequency of the comple- HIV shuttle 3.12 vpr ATG0 was used to test for comple- mented vector was not significantly different from the muta- mentation in trans of the vpr mutations made in HIV shut- tion frequency of the parental vector (x2 Å 0.23; P ú 0.1), tle vector 3.12. Complementation was determined based but was significantly different from the mutation frequency on the mutation rate of the vector. Step 2 cells containing of the vpr0 vector (x2 Å 11; P õ 0.01). This provides evi- HIV shuttle 3.12 vector vpr ATG0 were transfected with dence that a vpr0 vector can be complemented in trans the helper plasmids and the vpr expression plasmid. with a vpr expression plasmid.

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TABLE 2 Mutation Frequency in Recovered Proviruses of a HIV-1 Shuttle Vector Containing a Mutation in the vpr Gene That Limits Vpr Incorpo- ration into Virus Particles

No. of mutants/ Step 2 total No. of Mutation FIG. 4. Radioimmunoprecipitation analysis of Vpr expression and Mutant clone No. bacterial colonies frequency virion incorporation from Step 2 cells with HIV shuttle vector 3.12 or derivatives containing mutations in the vpr gene. Transiently 35 HIV shuttle 3.12 transfected Step 2 cells were metabolically labeled with [ S]Met; cell- 3 associated and virion-associated proteins were immunoprecipitated Vpr A30F 1 9/847 11 1 100 3 with a rabbit anti-Vpr serum. Lane 1, untransfected Step 2 cells with 2 24/1426 17 1 100 3 11/819 13 1003 the parental HIV shuttle 3.12; lane 2, parental HIV shuttle 3.12; lane 3, 1 0 Total 44/3092 14 1003 a HIV shuttle 3.12 vpr ATG ; lane 4, HIV shuttle 3.12 Vpr A30F; lane 5, 1 0 Parental, vpr/ HIV shuttle 3.12 vpr ATG complemented with vpr expression plasmid. HIV shuttle The positions of the molecular weight standards are shown on the 3 right. vector 3.12 1 4/1387 3 1 100 2 6/1063 6 1 1003 Total 10/2450 4 1 1003 Vpr was inefficiently expressed in nontransfected Step a Average mutation frequency with a standard deviation of 3 1 1003 2 cells from HIV shuttle 3.12 (Fig. 4A, lane 1) relative to mutants per cycle. Vpr in cells transiently transfected with the helper plas- mids and the tat expression plasmid (Fig. 4A, lane 2). The relatively low expression of Vpr from HIV shuttle 3.12 Expression of Vpr from virus-producing cells in the untransfected Step 2 cells provides an explanation The expression and virion-incorporation of Vpr with as to why these cells are able to proliferate in the pres- HIV shuttle 3.12 and derivatives used in this study was ence of the wild-type vpr gene. Vpr was detected in viri- analyzed (Fig. 4) to confirm the correlation between Vpr ons only from the transiently transfected cells producing virion incorporation and the influence of the vpr gene on virus particles (Fig. 4B, lane 2). In total, these data sup- the mutation rate. Expression of Vpr in cells with the vpr0 port the correlation between Vpr virion incorporation and mutant HIV shuttle 3.12 vpr ATG0 was not detected in the influence of vpr on the mutation rate. cells (Fig. 4A, lane 3) or virus particles (Fig. 4B, lane 3). Vpr was expressed with HIV shuttle 3.12 Vpr A30F in DISCUSSION cells (Fig. 4A, lanes 4), but was not detected with virus particles (Fig. 4B, lanes 4). In the trans-complementation The HIV-1 accessory gene vpr was mutated to deter- experiment of HIV shuttle 3.12 vpr ATG0 with a wild-type mine whether the vpr gene could, at least in part, account vpr expression plasmid, Vpr was detected from cells (Fig. for the lower than predicted in vivo mutation rate of HIV- 4A, lane 5) and from virus particles (Fig. 4B, lane 5). 1. Mutation of the vpr gene start codon was made and introduced into a HIV-1 shuttle vector that has been used to determine the in vivo mutation rate of HIV-1 (Mansky TABLE 3 and Temin, 1995). The mutation rate of this vpr0 vector Mutation Frequency in Recovered Proviruses of a vpr0 Shuttle was determined using the lacZa peptide gene as a re- 0 Vector Complemented in trans with a vpr Expression Plasmid porter for mutations. The mutation rate of a vpr HIV-1 was determined to be 12 1 1005 mutations per target No. of mutants/ base pair per cycle (Table 4). This mutation rate is four- Step 2 total No. of Mutation fold higher than that of the parental vector, 3 1 1005 Mutant clone No. bacterial colonies frequency mutations per target base pair per cycle (Table 4). Two HIV shuttle 3.12 vpr additional experiments supported the influence of vpr on ATG0 plus wild- the mutation rate of HIV-1. First, a HIV-1 shuttle vector type vpr plasmid 1 5/1041 5 1 1003 containing a mutation in the vpr gene that limits incorpo- 2 6/778 8 1 1003 03 ration of Vpr into virus particles had a mutation frequency 3 8/1173 7 1 10 0 Total 19/2992 6 1 1003 a similar to that of the vpr vector. Second, the mutation 0 Parental, vpr/ HIV frequency of the vpr vector was found to be comparable shuttle vector to the vpr/ parental vector when complemented in trans 3.12 1 4/967 4 1 1003 with a wild-type vpr expression plasmid. 03 2 7/1112 6 1 10 Mutation of the vpr gene did not significantly influence Total 11/2079 5 1 1003 the proportion of base-substitution or frameshift muta- a Average mutation frequency with a standard deviation of 1 1 1003 tions to the total number of mutants recovered, when mutants per cycle. compared to similar proportions of the parental vector

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TABLE 4 localization of HIV-1 DNA in nondividing cells (Heinzinger Relative Rate of Mutation for a HIV-1 vpr0 Mutant and a vpr/ HIV-1 et al., 1994). The importance of vpr in viral persistence and patho- No. of recovered mutant shuttle genesis is unclear. Reversion of an experimentally intro- vector proviruses duced mutation into the initiation codon of the SIV vpr gene was associated with disease progression (Lang et Mutation type HIV-1 vpr0 mutant vpr/ HIV-1 al., 1993); however, progression to AIDS can occur in the Base-pair substitution 33 16 absence of vpr (Gibbs et al., 1995). Nonsense mutations Frameshift 15 7 in the vpr gene have been characterized in isolates ob- Deletion 2 1 tained from HIV-1-seropositive individuals (Nakaya et al., Deletion with insertion 0 0 Overall mutation frequencya 50/3254 24/6462 1994), suggesting that virus persistence is associated Overall mutation rateb,c with the accumulation of nonsense mutations in the vpr (mutations/target bp/cycle) 12 1 1005 3 1 1005 gene. The in vivo mutation rate of HIV-1 is 20-fold lower than a Data from Table 1. the error rates of purified HIV-1 reverse transcriptase b The rates of mutation were calculated as the sums of the rates of base-pair substitution, frameshift, and deletion mutations per 3254 total with RNA and DNA templates of the lacZa peptide gene shuttle vector proviruses for a HIV-1 vpr0 mutant, per 6462 for vpr/ (Boyer et al., 1992; Mansky and Temin, 1995). Mutation HIV-1, per 114 target nucleotides for substitutions, per 150 target nucle- of the vpr gene leads to a HIV-1 mutation rate that is otides for frameshifts, or per 280 target nucleotides for deletion muta- fivefold lower than the in vitro error rates. This indicates tions. Target nucleotides for substitution and frameshift (Bebenek et al., 1989; Boyer et al., 1992) and deletion mutations (Pathak and Temin, that other protein factors may also influence the in vivo 1990b) have been previously described. mutation rate. The nucleocapsid protein, which is closely c To calculate the overall mutation rate, multiple mutants were put associated with the genomic RNA, is part of the viral into one of the mutation type categories based on mutations in each core particle, and is involved in the initiation of reverse mutant that occurred at known target nucleotides in the lacZa peptide transcription (Lapadat-Tapolsky et al., 1993), could also gene. Only one mutant in each multiple mutant was used in the calcula- tion of the rate. influence the accuracy of reverse transcription. Viral rep- lication accessory proteins that affect the accuracy of replication have been described for other viruses. For (Table 4). However, the increase in the overall mutation 0 example, bacteriophage T4 encodes two proteins, gene rate observed with the vpr vectors coincided with a 32 protein (a helix-destabilizing protein) (Hurley et al., similar increase in the rate of mutation of base-substitu- 1993) and gene 45 protein, that are part of the replication tion and frameshift mutations and a fourfold increase complex and have been shown to influence the accuracy in the number of mutants containing multiple mutations of replication (Mufti, 1979, 1980; Topal and Sinha, 1983; recovered to that of the parental vector. An increase in Watanabe and Goodman, 1978). the number of multiple mutants along with the increase The imbalance of deoxynucleotide triphosphate pools in mutation rate indicates that the incidence and not the has been suggested to influence the efficiency and accu- specificity of mutations occurring during the single round racy of HIV-1 replication and the fidelity of purified HIV- of HIV-1 replication was influenced by mutation of the 1 reverse transcriptase (Bebenek et al., 1992; Martinez vpr gene. et al., 1994; Meyerhans et al., 1994; Vartanian et al., 1994). The mechanism by which the vpr gene influences the mutation rate, as well as the function(s) of the vpr gene A possible effect of the vpr gene on HIV-1 reverse tran- in the replication cycle of HIV-1, is not entirely clear; the scription could be to influence the access of deoxy- influence of vpr on the mutation rate that was observed nucleotide triphosphates to reverse transcriptase. in this study could be either direct or indirect. However, our findings indicate that the vpr gene influences an early step in the replication cycle of HIV-1 (i.e., reverse tran- ACKNOWLEDGMENTS scription). The HIV-1 Vpr protein has been found to have several I am grateful for the guidance of Howard M. Temin during the initial stages of this work. I thank Xiao-Juan Bi, Eric Hausmann, Brad Seufzer, different influences and interactions. Vpr has been found and Rebecca Wisniewski for superior technical assistance; Donal to act intracellularly to influence productive infection and Kaehler for assistance in the McArdle BL-3 facility; Kathy Boris-Lawrie, latency (Levy et al., 1994, 1995), to influence HIV-1 tran- Dawn Burns, Norman Drinkwater, Eric Freed, Tom Mitchell, Gary Pulsi- scription (Cohen et al., 1990), to inhibit proliferation and nelli, Bill Sugden, and Shiaolan Yang for helpful discussions; Vinay activation of cell differentiation in a human muscle cell Pathak, Ron Swanstrom, and Alice Telesnitsky for stimulating conversa- line (Levy et al., 1993), to interact with cellular proteins tions; and Kathy Boris-Lawrie, Nito Panganiban, and Dan Loeb for dis- cussions and critical reading of the manuscript. This work was sup- (Bouhamdan et al., 1996; Refaeli et al., 1995; Zhao et al., ported by Grants CA22443 and CA07175 from the Public Health Service. 1994), to prevent cell proliferation during chronic infec- L.M.M. was supported by NRSA Viral Oncology Training Grant CA0975- tion (Rogel et al., 1995), and to be involved in the nuclear 17 and by a NIH Postdoctoral Fellowship.

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