Vol. 65, No. 11

Recombinational Analysis of a Natural Noncytopathic HumanImmunodeficiency Virus Type 1 (HIV-1) Isolate: Role of the vif

Gene in HIV-1 Infection Kinetics and CytopathicityKOJI SAKAI,1t XIAOYUE MA,' IRINA GORDIENKO,2 AND DAVID J. VOLSKYl*

Molecular Virology Laboratory, St. Luke'slRoosevelt Hospital Center, and College of Physicians and Surgeons,Columbia University, 432 West 58th Street, New York, New York 10019,1 and Yale University

School of Medicine, New Haven, Connecticut 065112

Received 19 March 1991/Accepted 25 July 1991

Two molecularly cloned coisolates of human immunodeficiency virus type 1 (HIV-1) have been found toexhibit different phenotypes of viral expression, either rapid and cytopathic (N1T-A virus) or delayed andnoncytopathic (N1T-E virus [X. Ma, K. Sakai, F. Sinangil, E. Golub, and D. J. Volsky, Virology 176:184-194,1990]). To identify the viral genetic elements responsible for these phenotypes, we prepared reciprocalrecombinants in different regions of N1T-A and N1T-E viral genomes. Infectivity experiments with therecombinant viruses revealed that the rapid/cytopathic (NlT-A-like) phenotype assorted cleanly with theVif-coding region and Vif expression. The smallest HIV-1 DNA region that conferred the complete phenotypicswitch was a 284-bp NdeI-StuI fragment within the vif open reading frame. Nucleotide sequence analysisrevealed a 35-bp deletion starting at nucleotide 218 in the N1T-E vif gene. A 23-kDa Vif protein was detectedby immunoblotting using Vif-specific antiserum in extracts of cells infected with N1T-A but not N1T-E virus.No detectable Vif protein was found in association with sedimented particles of either virus. Cotransfection ofa eucaryotic vif expression plasmid with N1T-E DNA complemented the N1T-E defect; rapid/cytopathicinfection similar to that in NlT-A-transfected cells was observed. We conclude that Vif controls the rate, andconsequently the cytopathic outcome, of HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) comprisesmany molecularly and functionally divergent isolates (re-viewed in references 7 and 44). HIV-1 infection in vitrodiffers in kinetics, productivity, and cytopathicity, depen-dent on both virus and cell type (1, 8, 10, 12, 19, 31, 32, 43,45). Similarly, the functional heterogeneity of HIV-1 mayplay a role in the outcome of HIV-1 infection in vivo, assuggested by the frequent isolation of highly cytopathicHIV-1 strains from patients at advanced stages of AIDS (1,8, 41, 43). Not surprisingly, the molecular basis of differentfunctional properties of HIV-1 has been a subject of inten-sive research (26, 27, 30, 32, 39).We are interested in the viral mechanisms of cytopathicity

and their control in vitro. We have previously described theisolation (3) and molecular cloning (32, 33) of a family ofHIV-1 isolates (HIV-1/N1T) from an individual with lymph-adenopathy. Among these viruses, N1T-A behaves likeprototypical HIV-1 isolates; its infection is rapid and cyto-pathic. N1T-E displays slow kinetics of infection and is notcytopathic, but like N1T-A, it is capable of efficient fusionand entry into target cells, expresses a transcriptionallyactive long terminal repeat (LTR) element, has functional tatand rev genes, and is highly productive in chronic infectionin T lymphocytes and monocytes (17, 27, 32, 33). Like othermultiple isolates from one individual (12, 19), N1T-E andN1T-A have similar restriction endonuclease maps and thusare closely related (32). The presence of relatively fewfunctional distinctions between these viruses makes the pair

* Corresponding author.t Present address: Laboratory of AIDS Epidemiology, AIDS

Research Center, National Institute of Health, 4-7-1 Gaku-en,Musashi-murayama-shi, Tokyo 190-12, Japan.

a good model for studies on the mechanisms controlling thecytopathic/noncytopathic phenotypes of HIV-1.One notable property of the noncytopathic N1T-E virus is

that despite its slow infection kinetics, its level of replicationeither in chronically infected cells or in transfectants of viralDNA is higher than that of its cytopathic counterpart,N1T-A (27, 33). This finding prompted us to evaluate thetranscriptional activities of the N1T-E and N1T-A LTRs(17). Consistent with the high replicative potential of theN1T-E virus, we found that the N1T-E LTR is significantlymore active than the N1T-A LTR in directing either viral orheterologous gene expression. However, the basal transcrip-tional activity of the LTRs did not correlate with either theviral infection kinetics or cytopathicity of the two clones(17). To identify the viral gene(s) which may control thesetwo functions, we constructed and functionally character-ized a series of reciprocal recombinants between protein-coding subgenomic fragments of N1T-A and N1T-E. Asso-ciation between infection phenotype and HIV-1 codingsequences of these viruses enabled us to map the regionresponsible for both the rapid kinetics of infection andcytopathicity to the vif gene. These results ascribe a novelfunction to the vif gene product in HIV-1 infection.

MATERIALS AND METHODS

Cell lines and recombinant vectors. The cells used includedCD4-positive cell lines of human T-cell leukemia origin CEM(14) and H-9 (16); HIV-1/IIIB-infected H-9/IIIB cells (15);and HIV-1/NlT-A- and HIV-1/NlT-E-infected CR10/NiT-Aand CEM/N1T-E cells, respectively (32). The H-9/IIIB cellculture was obtained from R. Gallo. The cells were main-tained in RPMI 1640 medium supplemented with 5% fetalbovine serum and antibiotics. The eucaryotic vif expression

5765

JOURNAL OF VIROLOGY, Nov. 1991, p. 5765-57730022-538X/91/115765-09$02.00/0Copyright © 1991, American Society for Microbiology

5766 SAKAI ET AL.

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FIG. 1. Schematic representation of recombinants between theN1T-A and N1T-E proviral DNA clones. Construction of therecombinants is described in Materials and Methods. NlT-A- andNlT-E-originated sequences are shown in open and filled boxes,respectively. Restriction enzyme sites: A, ApaI; E, EcoRI; N,NdeI; Sa, Sal; S, StuI.

vector, pNAL3 (40), was obtained from M. Martin. Itcontains vif cDNA derived from virus-infected H-9 cellscloned between the LTRs of the infectious molecular plas-mid pNL432 (40).

Construction of recombinant HIV-1 clones. Recombinantclones constructed between the N1T-A and N1T-E genomes

are shown schematically in Fig. 1. The plasmids used to

construct pNlT-A/E and pNlT-E/A were pNlT-A2 andpNlT-E2, which have proviruses and flanking cellular se-

quences cloned into pUC18 at XbaI sites in an orientationsuch that the Sall site of pUC18 is adjacent to the 5' XbaIsite of the insert (32). SaI fragments that contain the 5'halves of the virus clones were exchanged betweenpNlT-A2 and pNlT-E2 to yield pNlT-A/E and pNlT-E/A.KS series recombinants were constructed from pKS242 andpKS243, which carry N1T-A and N1T-E virus sequences,

respectively, with cellular flanking sequences shorter thanthose in pNlT-A2 and pNlT-E2. To construct pKS242, a

3.9-kbp PstI-XbaI 3' flanking cellular sequence was re-

moved, and the PstI end was flushed with T4 DNA polymer-ase and then ligated to a NotI linker (dGCGGCCGC). Theresulting 12.3-kbp XbaI-NotI fragment was inserted into the

XbaI-NotI sites of a pUC18-derivative plasmid in which the

PvuII site in the lacI' gene and SspI site were converted to

XbaI and NotI sites, respectively, by adding linkers (dCTCTAGAG and dGCGGCCGC). To construct pKS243, 3.0-kbpXbaI-EcoRI 5' and 1.4-kbp PstI-XbaI 3' flanking cellularsequences were removed; EcoRI and PstI ends were flushed

with T4 DNA polymerase and converted to XbaI and NotIsites, respectively, by linker additions. The resulting 11.8-kbp XbaI-NotI fragment was inserted into the same pUC18-derivative plasmid as described for pKS242. Throughout thisstudy, pKS242 and pKS243 were used instead of pN1T-A2and pNlT-E2 and are referred to as N1T-A and N1T-E in thetext. The biological activities of the viruses recovered frompKS242 and pKS243 are indistinguishable from those oforiginal N1T-A and N1T-E viruses recovered from pN1T-A2and pNlT-E2.Four pairs of reciprocal recombinants were constructed

from pKS242 and pKS243 by using EcoRI (yielding pKS262and pKS263), ApaI-plus-EcoRI (pKS274 and pKS275),XbaI-plus-ApaI (pKS278 and pKS279), and NdeI-plus-StuI(pKS282 and pKS283) digestions as shown in Fig. 1.

Evaluation of the biological activity of HIV-1 recombinants.Two methods were used to generate infectious progenyviruses for biological studies. In one method, 5 x 106adherent SW480 cells were transfected with 10 jig of plasmidDNA by the DEAE-dextran method (2), cultured in L-15medium for 48 h, and then cocultivated with 2 x 106 CEMcells. After 6 h of incubation at 37°C, the nonadherent cellswere transferred into RPMI 1640 medium supplemented with10% fetal bovine serum and antibiotics and cultured understandard conditions. At the indicated time points, cultureswere observed under a light microscope for the presence ofcytopathic effects, and aliquots of the cultures were testedfor cell viability and for expression of HIV-1 antigens as

detected by immunofluorescence (IF) and by HIV-1 p24antigen levels in culture supernatants. In the experimentshown in Table 1, each plasmid DNA was transfected intriplicate; 48 h later, HIV-1 p24 antigen levels were deter-mined in culture supernatants, and cultures showing compa-rable levels of HIV-1 production were chosen for cocultiva-tion with CEM cells. This allowed standardization ofprogeny virus titers used for infection. In an alternativeapproach, 5 x 106 CEM cells (for NlT-E-like viruses) or H-9cells (for NiT-A-like viruses) were transfected with 5 jig ofplasmid DNA by electroporation, using a Gene Pulser appa-ratus (Bio-Rad Laboratories, Richmond, Calif.) at 250 V and960 ,uF (time constant of 40 to 50 ms); 48 h later, 5 x 106uninfected CEM or H-9 cells were added to facilitate infec-tion, and cells were cultured under standard conditions untilmore than 90% of cells expressed HIV-1 antigens as detectedby IF. The long-term HIV-1 cultures established by thismethod were used for a large-scale production of recombi-nant progeny viruses.

Southern blot analysis of recombinant HIV-1 DNA. Totalcellular DNA was isolated from cells by the guanidiniumisothiocyanate method (2) at the time of maximum HIV-1-specific IF (days 3 to 7 for cells infected with N1T-A,NlT-A/E, KS263, KS274, KS278, and KS283; days 25 to 35for cells infected with N1T-E, KS275, and KS279; days 35 to45 for cells infected with NlT-E/A, KS262, and KS282).Total cellular DNA (10 to 20,ug) was digested either withHindIII or with NdeI plus StuI, subjected to electrophoresisthrough an agarose gel (0.8% for HindlIl digests and 1.5%for NdeI-plus-StuI digests), blotted onto a Nytran nylonmembrane (Schleicher & Schuell, Keene, N.H.), and ana-lyzed for HIV-1-specific sequences by hybridization. TheHIV-1 probes used for hybridizations were a 8.9-kbp SacIfragment derived from the N1G-G clone (42) and a 284-bpNdeI-StuI fragment from the N1T-A clone. The hybridizedfilter was washed three times for 30 min each at 50°C in 1 xSSC (0.15 M NaCl, 0.015 M sodium citrate)-1% sodiumdodecyl sulfate (SDS) before autoradiography.

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NONCYTOPATHIC HIV-1 ISOLATE 5767

Nucleotide sequencing. AvaII-NcoI fragments from theN1T-A and N1T-E clones were blunt ended with a Klenowfragment of DNA polymerase I and inserted into SmaI sitesof M13mp18 and M13mpl9. Single-stranded phage DNA ofthese M13 clones served as templates for sequencing by thedideoxyribonucleotide chain termination method (2) using aSequence kit (United States Biochemical Corp., Cleveland,Ohio). Deletion derivatives were constructed whenever nec-essary.Western immunoblot analysis of the Vif protein. Lysates

from virus-infected cells were prepared at the time of themaximum positivity for HIV-1-specific IF as noted above.Cells were washed twice with phosphate-buffered saline,resuspended at 5 x 107 cells per ml in 0.01 M Tris HCI (pH8.0)-0.14 M NaCl-1% Triton X-100-1 mM iodoacetamide-0.2 U of aprotinin per ml-1 mM phenylmethylsulfonyl fluo-ride-1% sodium deoxycholate-0.1% SDS, incubated for 1 hat 4°C, and centrifuged for 30 min in a microcentrifuge toremove cell debris. The culture supernatants were collectedat the same time, pelleted at 30,000 x g for 2 h, and lysed inthe same buffer. Fifty micrograms of total protein of eachlysate was loaded on a 10% SDS-polyacrylamide gel, elec-trophoresed, and blotted onto a Trans-Blot nitrocellulosefilter (Bio-Rad). The filter was incubated with rabbit antise-rum against synthetic peptides spanning from amino acids170 to 184 (TEDRWNKPQKTKGHR) of the Vif protein (agift of A. Adachi), washed, incubated with 125I-protein A,and autoradiographed.Complementation of the Vif- viral phenotype by vif expres-

sion vector. CEM cells (10 x 106) were transfected by theDEAE-dextran method (2) with 10 ,ug of pNlT-A, pNlT-E,or pNAL3 or with 5 jig of pNlT-E plus 5 ,ug of pNAL3.Transfected cells were cultured under standard conditionsand evaluated at the designated time intervals for cellviability (by trypan blue exclusion), cytopathic effects (bymicroscopy), expression of viral antigens (by IF), and levelsof viral p24 core antigen in culture supernatants (by p24assay). To determine cell-to-cell transmission of virus by thetransfectant cultures, cell samples were removed 2 daysafter transfection and cocultivated with uninfected CEMcells at a 1:10 ratio of transfectant to uninfected cells. Thecocultures were analyzed for HIV-1 expression and cyto-pathic effects as described above.

Other analytical procedures and reagents. Unless specifiedotherwise, plasmid propagation and DNA isolation, South-ern blotting, and other molecular biological procedures wereperformed according to standard methods (2). HIV-1 infec-tion was monitored as described previously (27, 32) by IFassay and by measuring the level of HIV-1 p24 capsidprotein in culture supernatants, using the Coulter HIV-1 AgAssay (Coulter Immunology, Hialeah, Fla.). Fluoresceinisothiocyanate-conjugated goat anti-human immunoglobulinG antibody [F(ab')2] was from Tago, Inc. (Burlingame,Calif.). Enzymes for recombinant DNA experiments werepurchased from New England BioLabs, Inc. (Beverly,Mass.), and Bethesda Research Laboratories (Gaithersburg,Md.); [35S]DNA and Rainbow protein molecular weightmarkers were from Amersham Corp. (Arlington Heights,Ill.), and other radiochemicals were obtained from NewEngland Nuclear Corp. (Boston, Mass.). Other chemicalswere purchased from Sigma Chemical Co. (St. Louis, Mo.).

RESULTS

Construction and analysis of recombinants between thecytopathic N1T-A and noncytopathic N1T-E coisolates. The

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FIG. 2. Southern blot analysis of viral DNAs in CEM cellsinfected with recombinant viruses. Total cellular DNAs from virus-infected cells were digested either with HindIII or with NdeI plusStuI, electrophoresed, and blotted onto nylon filters. The filterswere probed with a nearly full length (8.9-kbp) Sacl fragment ofHIV-1 DNA (probe A) for HindlIl digests (A) and with a 284-bpNdeI-StuI fragment of HIV-1 vif(probe B) for NdeI plus StuI digests(B). (C) Diagrams of the probes used. Numbers indicate kilobases.Abbreviations: H, Hindlll; (H), Hindlll present in N1T-E butabsent in N1T-A; N, NdeI; S, StuI.

recombinant NlT-E/NlT-A clones were constructed by per-forming reciprocal exchanges of subgenomic fragments be-tween the infectious molecular clones of N1T-A and N1T-Eviruses according to the schematic representation shown inFig. 1. To confirm that recombinant viruses consisted ofexpected genotypes, recombinant plasmids were examinedin detail, using 12 restriction enzymes that were used toanalyze the parental clones (32) as well as the enzymes usedfor recombination (data not shown). Then Hindlll-digestedtotal cellular DNAs isolated from recombinant virus-produc-ing cells were analyzed for HIV-1-specific sequences by theSouthern blotting method, using a nearly full length (8.9-kbp)HIV-1 DNA as a probe (probe A; Fig. 2C). As expected,

VOL. 65, 1991

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5768 SAKAI ET AL.

TABLE 1. Biological activities of the recombinants between N1T-A and N1T-E viruses

Results obtained at:

Virus clone Day 0, Day 3 Day 5 Day 10 Day 15inputp24a Viab IF" CPEd p24e Via IF CPE p24 (pg/ Via IF CPE p24 (pg/ Via IF CPE p24 (pg/

(pg/ml) (%) (%) (pg/ml) (%) (%) ml) (%) (%) ml) (%) (%) ml)

N1T-A 278 85 6 + 5,700 74 81 ++ 385,000 24 75 ++++ 425,000 20 73 +++ 206,500N1T-E 317 94 <0.1 - 580 92 0.1 - 1000 98 2 - 1963 86 1.5 - 950N1T-A/E 400 88 20 +++ 37,000 43 82 ++++ 520,000 2 62 ++++ 117,000 NAf NA NA NAN1T-E/A 314 90 <0.1 - 720 91 0.5 - 660 89 0.1 - 910 86 0.5 - 240KS262 166 89 <0.1 - 230 94 0.1 - 130 94 0.1 - 296 85 0.2 - 86KS263 234 91 7 + + 8,000 59 81 +++ 965,000 44 69 ++++ 200,000 44 67 +++ 7,000KS274 67 80 1 + 2,000 83 65 + + 170,000 25 53 +++ 190,000 23 71 ++++ 48,000KS275 300 86 <0.1 - 190 90 0.1 - 160 93 0.1 - 940 92 1.4 - 1,180KS278 279 90 3 + 1,800 76 53 + + 330,000 41 70 +++ 285,000 22 62 + + + + 100,700KS279 403 88 <0.1 - 560 90 0.5 - 850 92 0.7 - 50 89 5 - 4,300KS282 118 93 <0.1 - 90 95 0.1 - 382 94 0.1 - 50 93 0.1 - 10KS283 298 85 9 + 18,500 62 77 +++ 600,000 45 77 ++++ 290,000 2 53 +++ 95,000

a HIV-1 p24 measurement in cell-free culture supematant 48 h after SW480 cell transfection.b Via, cell viability determined by trypan blue exclusion.c IF, HIV-1-specific antigen determined by indirect IF assay.d CPE, cytopathic effects by the observation of giant cells encompassing less than 1% (+) to 30% or more (+ + + +) of the total cell population.e HIV-1 p24 antigen in cell-free culture supernatant.f NA, not applicable; observations or measurements could not be made.

8.0-kbp bands were detected with viruses bearing N1T-Aenv (Fig. 2A, lanes 1, 4, 5, 7, 9, and 11), and two bands (6.5and 1.5 kbp) were detected with those carrying N1T-E env(Fig. 2A, lanes 2, 3, 6, 8, 10, and 12) due to an HindIII sitepresent in the env gene of the N1T-E viral genome as shownin Fig. 2.

Biological activities of recombinant viruses. The N1T-Avirus exhibits infection characteristics of a typical HIV-1,including rapid kinetics of infection and cytopathicity, andN1T-E infects cells slowly and without cytopathic effects;both viruses replicate efficiently in chronically infected cells(27, 32, 33). The recombinant progeny viruses clearly segre-gated into the two parental phenotypes of virus expression:NlT-A-like or NlT-E-like (Table 1). Evaluation of the initialpair of recombinants (NlT-A/E and NlT-E/A) demonstratedthat the slow kinetics/low cytopathicity phenotype is con-trolled by the 5' half of the N1T-E viral genome (Table 1).The NiT-A/E recombinant was similar to N1T-A virus inoverall characteristics (Table 1), showing that the 3' half ofN1T-E functions like that of cytopathic N1T-A virus. Ourprevious data also showed that N1T-E exhibits normalbinding and entry but delayed viral expression (27).

Evaluation of recombinants containing reciprocal ex-changes of 5' LTR-plus-gag, pol, vif-plus-vpr, and vif generegions revealed that the phenotypic switch between N1T-Eand N1T-A maps exclusively to the 284-bp NdeI-StuI frag-ment within the vifopen reading frame (Table 1). The resultsof a representative experiment of three performed areshown. The experimental format, involving the evaluation ofmultiple pairs of recombinants as shown in Table 1, was usedpredominantly to evaluate the switch from the slow/noncy-topathic (NlT-E-like) to fast/cytopathic (NlT-A-like) pheno-type. The vif-dependent phenotypic switch between N1T-Aand N1T-E (fast/cytopathic to slow/noncytopathic) and viceversa was confirmed by detailed examination of the infectioncharacteristics of KS282 and KS283 produced in long-termcultures (Fig. 3). pKS282 contains the viffragment from theN1T-E clone, and exposure of CEM cells to concentratedvirus at a multiplicity of infection of 1 yielded the NlT-E-like(slow/noncytopathic) infection phenotype (Fig. 3). The re-

ciprocal fragment exchange, in plasmid pKS283, yielded thefast/cytopathic NlT-A-like phenotype. Notably, the KS282virus had slower infection kinetics than did N1T-E virustested under the same conditions, consistent with the factthat the KS282 virus carries the N1T-A LTR, which is aboutthree times less efficient than the N1T-E LTR in directingHIV-1 expression (17).The noncytopathic N1T-E virus is a natural deletion mutant

in the vif gene. Comparative nucleotide sequence analysis ofthe AvaII-NcoI Vif-coding regions in N1T-A and N1T-Erevealed that N1T-E virus contains a 35-base deletion start-ing at nucleotide 218 (Fig. 4). This deletion causes a frame-shift in NIT-E vif coding sequence, resulting in a prematuretranslational termination at nucleotide 254 in the N1T-Esequence. Thus, N1T-E is a naturally occurring deletionmutant in the vif gene. This deletion was confirmed bySouthern blot analysis of the recombinant virus DNAsisolated from virus-producing cells. A Southern blot of totalcellular DNAs digested with NdeI plus StuI was probed witha 284-bp NdeI-StuI fragment of the N1T-A clone (probe B;Fig. 2C). As expected, recombinant viruses carrying theN1T-E vif gene generated fragments (presumably 249 bplong; Fig. 2B, lanes 2, 4, 5, 8, 10, and 11) shorter than thosefrom N1T-A vif-carrying viruses (Fig. 2B, lanes 1, 3, 6, 7, 9,and 12).Sequence analysis also showed a G-to-A mutation at

nucleotide 639 in the N1T-E clone which generated a termi-nation codon (TAG) in the vpr coding region. This mutationpresumably produces a 17-amino-acid truncated Vpr pro-tein. Since this mutation is outside the NdeI-StuI fragmentwhich confers the phenotypic switch between the N1T-Eand N1T-A viruses, it is not relevant to the biologic func-tions studied here.

Vif protein is present predominantly in extracts of infectedcells. Cells chronically infected with KS282, KS283, N1T-A,or N1T-E were evaluated for HIV-1 Vif protein by immuno-blotting, using antiserum specific for a synthetic Vif peptidefragment (Fig. 5). As predicted, a 23-kDa protein wasdetected with the Vif-specific antiserum in extracts fromN1T-A- and KS283-infected cells (Fig. 5, lanes 2 and 4) but

J. VIROL.

NONCYTOPATHIC HIV-1 ISOLATE 5769

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FIG. 3. Biological activities of KS282 and KS283 vif recombi-nant viruses during extended culture. CEM cells (2 x 106) were

infected with concentrated cell-free viruses (106 pg of HIV-1 p24protein) and cultured in RPMI 1640 medium; at the indicated timepoints, aliquots of the culture fluid were tested for cytopathicity(indicated by cell viability) and virus replication (HIV-i-specific IFof acetone-fixed cells and level of HIV-1 p24 antigen in cell-freeculture supernatant).

not from N1T-E and KS282 carriers (Fig. 5, lanes 3 and 5).No Vif-specific reaction was observed in immunoblots ofconcentrated virions (Fig. 5, lanes 7 and 9), suggesting thatmost Vif protein produced in chronic infection assorts tointracellular compartments rather than cell-free virus parti-cles, which may contain small amounts of Vif undetectableby this method.Complementation of vif defect in N1T-E by independent

endogenous expression of Vif. To confirm that the slow/cytopathic phenotype of N1T-E virus infection is due to thevif mutation and the absence of Vif expression, we testedwhether N1T-E virus infection can be modified in trans bycoexpression of Vif in the same cells (Fig. 6). CEM cellswere cotransfected with pN1T-E and the eucaryotic vifexpression vector pNAL3 (40). The infectious proviral DNAclones pN1T-A and pN1T-E were used as the wild-type andvif mutant-like infection controls, respectively. Kinetics of

AvallN1T-A GGACCAGCAA ACCTCCTCTG GAAAGOTGAA GGGGCACTAG TAATACAAGA TAATACTGAC ATAAAACTAC 70N1T-E ---------- ---------- --------- -70

e x v Q v I v V Q v 13N1T-A TGCCAAGAAG AAAAGCAAAG ATCATrAGGC ATrATGGAAA ACAGATOCA GCTCATGATT CTGTGGCAAG 140

N1T-e ---------- ---------- ---------- ---------- ---------- ---------- ---------- 140M N A V Q V I V V Q V 13

<- polNdeiD m x z R r V x S L V N r V S C A X 36

N1T-A TAGACROGAT GAGGATTAGA ACATCGAAAA CTrVAGTAAA ACACCATATC TATCTTTCAG GGAAAGCTAG 210N1T-E ---------- ---------- ---------- ---------- ---------- ---------- ---------- 210

D x m x I R r V x S L V X m r V S C X A R 36

G V F x YEr S PU?8 x I S S I V I P L 59N1T-A GGGATGOTr TATAGACATC ACTATGAAAC CCCTCATCCA AGAATAACTT CAGAACTACA CATCCCACTA 280N1T-E -------was sumussu *mmsmm *ssussuss on-------- ---------- ---------- 245

C V N F X S T H P T R 48

C D A R L V I r r V G L N C E X O V N L C Q 83NIT-A GGGGATGCTA GATrGGTAAT AACAACATAT TCOGCTCTGC ATACAGGAA AAGAGACTGG CATTTGCCTC 350

N1T-E ---------- ---------- ---------- ---------- ---------- ---------- ---------- 315C C * 50

G V S I V R K R Y S Q V D P E L A D Q L 106N1T-A AGGGACTCTC CATAAAATGG AGGAAAAA GATATAGCAC ACAACTAGAC CCTGAACTAC CAGACCAACT 420N1T-E - -- -- - - - - - --------- ------- ---------- -------------------G---------A. 385

I L Y Y F O C F S D S A I X A L L C I V 129N1T-A AATTCATCTC TATTACTTTG ACTGTTTTC AGACTCTCT ATAAGAAAGG CCTTATTAGG ACACATACTT 490

N1T-e -------- --------- ---------- --------- -------------------- -------- 455A 3asj

S P R C E Y Q A CM N V C S L Q Y L A L A A L 153N1T-A ACCCCTAGGT CTGAATATCA ACCAGGACAT AACAAGGTAG GATCTCTACA ATACTYGCCA CTACCAGCAT 560NIT-E ---------- ---------- ---------- -------- ---------- ---------- ---------- 525

5'uj A

vpr ->

I T P K I P P L P S V L E D R V X 176NIT-A TAATAACACC AAAAAGATA AACCCACCTr TGCCTAGTCT TACGAAACTC ACAGAGGACA GATGGAACAA 630N1T-E ---------- -----A-----C-------- ---------- ---T--------------T- ---------- 595

P Q X r X G o R R S O r J N C N * 192N1T-A CCCCCAGAAG ACCAAGGGCC ACAGAAGGAG CCACACAATG AATGACACT AGACTTTTA GAGGAGCTTA 700N1T-E ---------- ---------- -----C---- ---------- ---A------ ---------- ---------- 665

l4oIcN1T-A ACAATGAAGC TCTrACACAT TCTCCTACGA TTTCCCTCCA TCC 743NT-E ---------- ---------- -T-------- ---------- --- 708

FIG. 4. Aligned nucleotide sequences of the vif gene regions ofthe N1T-A and N1T-E clones. Deduced Vif amino acid sequencesand position numbers are shown in italics above (N1T-A) and below(N1T-E) the corresponding nucleotide sequences. In the N1T-Esequence, a dash indicates a base identical to that in N1T-A and abox denotes a gap. Asterisks in the amino acid sequences representtranslational termination sites.

HIV-1 expression and cytopathic effects were monitored intransfected cells and in cocultures of the transfectants withuninfected CEM cells.As shown in Fig. 6A, rapid/cytopathic infection developed

in CEM cells within 8 to 11 days after transfection withpN1T-A DNA; over 95% of the transfectants expressedHIV-1-specific antigens, and the level of virus productionreached 390 ng of extracellular HIV-1 p24 antigen per ml byday 11. Cells transfected in parallel with the same amount ofpN1T-E DNA showed little evidence of virus production byday 11, and no cytopathic effects were observed. Theslow/noncytopathic infection in N1T-E transfectants was notdue to inefficient DNA transfection, because 2 days post-transfection, pNlT-A- and pNlT-E-transfected CEM cellshad comparable proportions of IF-positive cells (2.3%; Fig.6A) and produced similar levels of HIV-1 p24 core antigen(267 and 700 pg/ml of supernatant, respectively). Cotrans-fection ofCEM cells with pN1T-E and pNAL3 resulted inN1T-A virus-like infection, including fast kinetics of HIV-1antigen expression, high virus production level, and cyto-pathicity (Fig. 6A). Thus, in cotransfection, the N1T-Edefect was fully complemented by the pNAL3 clone. Trans-fection of the pNAL3 DNA alone yielded no detectableeffects, notably no cytotoxicity.To determine whether complementation of N1T-E by

pNAL3 involved increased cell-to-cell transmission, sam-

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5770 SAKAI ET AL.

A~A,'11~ri, A ~ACJU~~CC)Z & 1,t+A\t?

2 3 4 5 6 7 8 9 10 11 12 13 14 15kDa

4_ 4- w- * 97.4

- 69

- 46

- 30

Vif .(23 kDa) - 21.5

- 14.3

Rabbit Anti-Vif Control Rabbit9 Serum-- -- Serum--

FIG. 5. Western blot analysis of the Vif protein. Cell lysateswere prepared from virus-producing cells at the time of the maxi-mum positivity of HIV-1-specific IF as described in Materials andMethods. Virus particles were pelleted from culture supernatantscollected at the same time. Fifty micrograms of total protein of eachlysate was loaded on each lane. A blotted nitrocellulose filter wasincubated with rabbit antiserum against synthetic peptides spanningfrom amino acids 170 to 184 (TEDRWNKPQKTKGHR) of the Vifprotein. Positions of molecular weight markers are shown on theright.

ples of the transfectants depicted in Fig. 6A were withdrawn2 days after transfection, washed, and cocultivated withCEM cells at a 1:10 cell number ratio (Fig. 6B). Cellschronically infected with N1T-A and N1T-E virus served asthe wild-type and vif mutant cocultivation controls, respec-tively. As expected, rapid/cytopathic infection was observedin cocultures of either pN1T-A transfectants or NlT-A-infected cells, reaching peaks at 9 and 4 days, respectively.Consistent with previously published results (27), slow/noncytopathic infection was observed in cocultures of N1T-E-infected cells. The pN1T-E-plus-pNAL3 transfectants,which by themselves expressed and replicated virus effi-ciently (Fig. 6A), were unable to spread viral infection incoculture with uninfected cells; neither significant virusreplication nor expansion of the number of HIV-1-positivecells was detected during 9 days of observation (Fig. 6B).This result indicates that complementation of N1T-E bypNAL3 is limited to cotransfected cells and that the infec-tious viral particles generated in these cells remain defectivefor cell-to-cell transmission, similar to the parental N1T-Evirus (27) and some (11) but not all (40) vifmutants describedby other laboratories.

DISCUSSIONWe have identified vif as the gene responsible for the

fast/cytopathic phenotype of HIV-1 infection. The N1T-Evirus has a 35-bp deletion in vif which prevents the expres-sion of intact Vif protein. The vif mutation is the sole defectresponsible for the slow/noncytopathic phenotype of theN1T-E virus because replacement with the analogous 284-bpvif fragment from the N1T-A virus completely reverses thephenotype of viral expression, notably including the synthe-

sis of full-length Vif protein. Thus, the cytopathic function ofN1T-A virus is intrinsically linked to the rapid rate of viralexpression, and both of these functions require a functionalHIV-1 vif gene.

Previous studies have shown that molecular clones madedeficient in the vif gene have diminished infectivity andlimited capacity to establish stable infection (11, 40). Someof these mutants can spread efficiently by cocultivation (40);others cannot (11). These data prompted the designation ofVif as a factor required for viral infectivity (11, 20, 40). Ourresults qualify this designation in several respects.

(i) HIV-1 can establish stable infection in the absence ofthe 23-kDa vif product. Infection with the N1T-E virus andKS282 yield cultures which are more than 90% IF positiveand produce high titers of infectious virus (Fig. 3) butcontain no 23-kDa vif product detectable by immunoblotting(Fig. 5). Thus, in the case of the N1T-E/N1T-A pair ofviruses, a full-length Vif is not required for viral infectivityper se. These results differ from the previous findings ofStrebel et al. (40) and Fisher et al. (11), who reported that vifmutants replicate poorly from viral DNA and cannot estab-lish stable infection. Several mechanisms may contribute tothe more robust infectivity of the N1T-E virus. The smallsize and location of the deletion in vif (35 bp, starting atnucleotide 218) minimally disrupt the HIV-1 RNA splicingevents which utilize this region of the HIV-1 genome (the 3'and 5' splice junctions are about 200 nucleotides down-stream of the deletion in the vif gene; Fig. 4 [28]). Alterna-tively, the differences may reflect the intrinsic structural andfunctional heterogeneity among different HIV-1 isolates (1,8, 13, 19, 27, 32, 41). Because the N1T-E virus LTR hasbasal transcriptional activity higher than that of the N1T-Aor SF-3 LTR (17), it may compensate for the initial lowlevels of N1T-E DNA, thus facilitating the establishment ofstable infection. Finally, truncated Vif (Fig. 4), which is notrecognized by the antiserum used, if partially functionalcould contribute to the observed N1T-E phenotype. Nota-bly, vif mutants 6.9 and 3.3, which were designed to produceVif proteins lacking carboxy-terminal residues similar to thepredicted N1T-E vifproduct, were incapable of stable infec-tion (11). This suggests that other mechanisms, in addition tolack of Vif expression, limit the infectivity of these mutants.

(ii) Vif is required for rapid infection with HIV-1. Sincevif* viruses infect cells faster than their vif mutant counter-parts (Fig. 3 [11, 40]), Vif function may be required toestablish conditions necessary for the typically rapid HIV-1infection in vitro. Notably, virus binding and entry intoCD4-expressing cells, functions controlled by the fusogenicEnv glycoprotein of HIV-1 (22, 35, 38), are not affected bythe vif mutation, because N1T-E virus has intact virus-cellfusion activity (27) and the env gene is outside the region thatconfers the phenotypic switch between N1T-E and N1T-A(this work). Similar amounts of viral nucleocapsids areinternalized during N1T-A or N1T-E infection, but N1T-Einfection is retarded in the synthesis of viral RNA andprotein (27) as well as of viral DNA (27a). One explanation ofthese results is that vif mutants have a defect at an earlystage of the viral life cycle after viral entry but before viralDNA synthesis, resulting in inefficient processing of viralnucleocapsids. Both primary infection and reinfection withprogeny virions would be affected, thus contributing to theobserved slow phenotype ofN1T-E virus. It is noteworthy inthis context that recent work implicated Vif as an enzymewhich could cleave Env in the inner membrane-facing do-main of gp4i (18). This cleavage may be required during

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NONCYTOPATHIC HIV-1 ISOLATE 5771

A

HIV-1 expression and cytopathiceffects 11 days post-transfection

Transfected DNA CPE p24 (ng/mI)

-U- pNlT-A +++ 390

-4-- pNlT-E - 6

pNIT-E + pNAL3 + + 210

--&- pNAL3 - 0

4 8 12Days after transfection

BHIV-1 expression and cytopathiceffects 9 days post co-cultivation

Transfected cells usedfor co-cultivaton t ] CPE p24 (ng/ml)

[NIT-A] +++ 379

[+NIT-E] - 0.08

[NIT-E + NAL3] ± 25

[NAL3] - 0

CR10/A* ++++ 495

CEM/E + 63

5

Days after co-cultivation

10

FIG. 6. Complementation of the N1T-E virus defect with a vif expression vector. CEM cells were transfected with the designatedplasmids, and transfectants were evaluated for HIV-1 expression and cytopathicity as described in Materials and Methods. (A) Analysis ofthe kinetics of virus expression (graph) and cytopathic effects (table) in transfected cells; (B) kinetic analysis of virus expression (graph) andcytopathicity (table) in cocultures of CEM cells with the transfectants used for panel A. For cocultivation, transfectants were removed 2 daysafter transfection; the supernatant p24 levels at this time (in picograms per milliliter) were as follows: pNlT-A, 267; pNlT-E, 700; pNlT-Eplus pNAL3, 687; and pNAL3, 0. CR10/A* and CEM/E* represent cocultures of uninfected CEM cells with chronically infected CR10/N1T-Aand CEM/N1T-E cells, respectively (33); the cells were >90% positive for HIV-1 antigens by IF at the time of cocultivation. Cytopathic effect(CPE) of + + + + characterizes a culture in which >50% of cells were dead as determined by trypan blue exclusion and >50% of cells formedgiant cells of three or more.

virion assembly to ensure efficient processing of the inputvirus during subsequent infection.

(iii) Role of Vif in cell-to-cell transmission. Similar to thevif mutants described by Fisher et al. (11), the NlT-E-likeviruses exhibit defective cell-to-cell transmission duringcocultivation of acceptor cells with chronically N1T-E in-fected cells (27) or with N1T-E DNA transfectants (Fig. 6[27]). In both situations, virus replicates well and viralenvelope glycoproteins are present on cell membranes andmediate cell-to-cell fusion (27), yet viral infection spreadsslowly. These results suggest that a functional Vif proteinmay also be required for the cell-to-cell transmission of viralgenetic information or for its immediate expression, and thatdefects in these processes may retard infection with the vif

mutant viruses. A virus-encoded protein which plays a rolein cell-to-cell transmission of plant viruses has been recentlydescribed (5).

(iv) Correlation between vif gene expression and cyto-pathicity. Several recent reports described HIV-1 or HIV-2isolates with attenuated cytopathicity (4, 9, 10, 13, 23, 39,41). For the majority of these isolates, the noncytopathicphenotype has been attributed to defects in HIV envelopeglycoproteins (4, 13, 39). However, in the virus pair studiedhere, replacement of the vifgene in N1T-A virus with N1T-Evif is sufficient to convert a previously cytopathic (and thusEnv functional) virus to a noncytopathic phenotype (Table 1and Fig. 3). Accelerating N1T-E virus expression by supply-ing functional vif (Fig. 4 and 6) may facilitate the critical

> 100

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VOL. 65, 1991

5772 SAKAI ET AL.

accumulation of viral (21, 25, 34, 35, 37) and cellular (29)products necessary to achieve the cytopathic effect of thevirus. These results suggest a novel requirement for HIV-1cytopathicity involving rapid kinetics of infection.

Implications. The results of this analysis underscore thecomplexity of the HIV-1 life cycle and its regulation. N1T-Evirus exhibits both high basal replicative activity in stableinfection or after viral DNA transfection because of its"fast" LTR (17) and slow primary infection kinetics becauseof defunct vifgene (this work). Through its effect on the rateof HIV-1 infection (but not the establishment of stable virusproducing-cell lines), vif modifies a distinct viral function,namely, cytopathicity. In the recently described mf-D viralclone from the same NiT stock (6), cytopathicity is affectedby mutations in the env gene (39). It is unclear whether thesemutations arose in vivo or during molecular cloning of mf-D.Our ongoing analysis of the virus extant in the individualfrom whom the NiT isolate was obtained in 1984 (3) willresolve this question. These data indicate that similar viralphenotypes can arise by distinct molecular mechanisms.This diversity must be considered in devising strategies tocontrol HIV-1 infection.

ACKNOWLEDGMENTS

We acknowledge N. S. Hamblet and X. P. Ma for technical help,A. Adachi for rabbit anti-Vif serum, R. Gallo for the H-9/IIIB cellline, M. Martin for the pNAL3 vector, and L. Ngai for typing themanuscript. Thanks are extended to M. J. Potash for stimulatingdiscussions and editing the manuscript.

This work was supported in part by grants CA37465, A125902, andA127397.

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