AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 11, Number 7, 1995Mary Ann Liebert, Inc.

Short Communication

Genetic Construction and in Vitro Characterization ofSIVsmmPBj 14-1.9 Noninfectious Particles

MARGUERITE DESCHAMPS,1 BENEDICTE LAMBRECHT,1 MARIE HORTH,1 SUZY KÜMMERT,2HANS R. GELDERBLOM,3 CLAUDINE BRÜCK,2 and ARSENE BURNY1

The human retrovirus HIV (human immunodeficiencyvirus) is the etiological agent of the acquired immunode-

ficiency syndrome (AIDS). Simian immunodeficiency virus(SIV) and HIV are homologous in their biological and physi-cal properties and in their genomic structure.1 Unlike other SIVisolates, which induce a syndrome in experimentally infectedmacaques that is remarkably similar to human AIDS,2,3 theSrVsmmPBjl4 isolate causes an acute disease characterized byhigh titers of virus in the blood and lymphoid tissues as wellas elevated levels of acute-phase inflammatory reactants andcytokines leading to the death of pigtail macaques and othermonkey species within 6 to 8 days.4-7 Thus, the SIVsmmPBj-14—macaque animal model system permits rapid evaluation ofthe efficacy of candidate vaccines. The molecular cloneSrVsmmPBjl4-1.98 (1.9; see Table 1 for a summary of thenomenclature) of SIVsmmPBj 14 is also pathogenic when in-oculated into pigtail macaques and induces, 7 days postinocu-lation, an acute infection that is characterized by marked cell-associated and cell-free viremia and a reduction in the CD4+and CD8+ T lymphocyte populations.9 However, unlike pigtailmacaques infected with the uncloned SIVsmmPBj 14, animalsinfected with the molecularly cloned 1.9 virus survive the acute-

phase disease and enter an asymptomatic phase of infection.1,9The aim of this study was to determine the conditions

whereby genetically inactivating the replicating functions of the1.9 proviral genome using site-directed mutagenesis producesstructurally intact noninfectious 1.9 mutant virions in order totest either the particles or the proviral DNA as a candidate vac-

cine. The pathogenic 1.9 molecular clone was selected amongexisting molecular clones of SIVsmmPBj 14 to create a situa-tion similar to that encountered with pathogenic HIV,SIVsmmPBj 14 being foreseen as the challenge virus. Using theplasmid SIVPBjl.9 (pl.9-wt, Table 1 and Fig. 1) (kindly pro-vided by S. Dewhurst, University of Rochester, NY),8 four plas-

mids deleted of their 3' long terminal repeat (LTR), with or

without the additional deletion of the overlapping nef'gene, were

constructed in an attempt to inactivate the replication and inte-gration functions of the virus produced while keeping it struc-

turally intact. At the level of replication, it was anticipated thatdeletion of the 3' LTR would produce noninfectious particlesbecause the 3' LTR, as well as the 5'LTR, are essential for re-

verse transcription and integration. 10-12 Furthermore, the nefgene, which partially overlaps the 3' LTR (Fig. 1), has alsobeen shown to play an important role in in vivo viral replica-tion.13,14 To potentially improve the level of viral productionthe incomplete 5' LTR present in the original pl.9-wt plasmidwas reconstituted because the missing upstream sequences ofthe incomplete 5 ' LTR are thought to be involved in the sub-tle up- and/or downregulation of viral expression. In particular,the well-characterized regulatory sequence NRE,15 covering a

large portion of the upstream 5 ' LTR region, has been associ-ated with the inhibition of viral transcription.16-19 In contrast,it has also been suggested that the NRE is an indirect target forthe nef gene product,17,20 whose role as a transcription regula-tor still remains controversial.20-23 On the basis of these data,the 3' LTR-deleted mutant viruses were produced under thecontrol of the incomplete or full-length 5' LTR in the presenceor absence of the nef gene.

The 3' LTR, also containing a major part of the nef gene se-

quence, was deleted and replaced with the exogenous polyadeny-lation region of bovine growth hormone (BGH-terminator)24,25in order to ensure correct messenger RNA translation termina-tion, normally controlled by the R/U5 region of the 3' LTR.26,27The nefgene was kept in two of the four mutant proviral DNAs.The full-length 5' LTR was constructed by duplicating its miss-ing sequences from the U3 present in the 3' LTR of pl.9-wt andinserting the newly obtained fragment at the 5' end of the 1.9proviral genome. Finally, the simian virus 40 (SV40) origin of

'Laboratory of Biological Chemistry, Free University of Brussels, rue des Chevaux, 67, 1640 Rhode-St-Genese, Belgium.2Department of Molecular and Cellular Biology, SmithKline Beecham Biologicals, rue de l'Institut, 89, 1330 Rixensait, Belgium.3Robert Koch-Institut, Nordufer 20, D-13353 Berlin 65, Germany.

855

856 DESCHAMPS ET AL.

Table 1. Nomenclature of Native and MutantSIVsmmPBjl4-1.9 Viruses and Plasmids"

A)

Molecular clone Virus Plasmid

SIVsmmPBjl4-1.98 1.9 pl.9-wt.SIVsmmPBj 14-1.9-ori 1.9-ori pl.9-oriSIVsmmPBjl4-1.9-oriC 1.9-oriC pl.9-oriCSIVsmmPBj 14-1.9-env 1.9-env pl.9-oriCSIVsmmPBjl4-1.9-envC 1.9-envC pl.9-envCSIVsmmPBj 14-1.9-nef 1.9-nef pl.9-nefCSIVsmmPBj 14- 1.9-nefC 1.9-nefC pl.9-nefC

"Native virases group 1.9, -ori, and -oriC. Mutant virusesgroup 1.9-env, -envC, -nef, and -nefC.

replication (SV40 ori) was cloned into each plasmid (with theexception of pi .9-wt) in order to assess transient 1.9 particle ex-

pression in COS-1 cells. The genetically altered regions presentin each plasmid was verified by restriction enzyme analysis, se-

quence analysis, and polymerase chain reaction (PCR) analysis(details of constructions are available on request).

Thus, we engineered six plasmids derived from pi.9-wt (Fig.IB: 0) (Table 1). pl.9-ori and pl.9-oriC contain the wild-type1.9 proviral genome with an incomplete or complete 5' LTR,respectively (Fig. IB: 1 and 4). Mutant plasmids pi.9-env andpl.9-envC are nef and 3' LTR-deleted plasmids containing an

incomplete and complete 5' LTR, respectively (Fig. IB: 2 and5). Mutant plasmids pl.9-nef and pl.9-nefC are 3' LTR-deletedbut not ne/-deleted plasmids, containing an incomplete andcomplete 5' LTR, respectively (Fig. IB: 3 and 6). The mutant

plasmids were named after the last retained gene at the 3' endof the proviral genome. For the purpose of this article, pi .9-wt,p 1.9-ori, and pl.9-oriC are collectively referred to as nativeplasmids.

The expression and maturation of particles produced aftertransient transfection of the native and mutated 1.9 plasmidsinto COS-1 cells demonstrated that these mutant particles are

similar to the native virus (Fig. 2). Indeed, immunoprecipita-tion with a 1.9-specific antiseram of radiolabeled SIV antigensfrom ultracentrifuged supernatants or cellular lysates of thetransfected COS-1 cells showed that the mutations do not alterthe expression or processing of the Gag precursor (p55) or theEnv precursor (gpl60). p55 and gpl60 were mostly present inthe cellular lysate, whereas the processed products (p24 andgpl20, respectively) were detected mainly in ultracentrifugedculture supernatants. The presence of reverse transcriptase (RT)activity (data not shown) in addition to Gag and Env in the ul-tracentrifuged supernatants strongly suggested that the viralproteins are associated with SIV virions. The fainter signal ofpi.9-wt was probably due to its lack of an SV40 ori, thus pre-venting it from replicating to a high copy number in the trans-fected COS-1 cells.28

To assess the infectivity of the mutant 1.9 viruses, both mu-

tant and native plasmids were transfected in two different tran-sient expression systems: lymphoid CEMxl7429 cells and COS-1 cells (Fig. 3).29a-c Transfection of all plasmids into CEMxl74was performed to subsequently evaluate the infectivity of thevirus particles produced in a lymphoid milieu. The higher lev-els of virus production obtained on transfection of native andmutant plasmids in COS-1 cells made it possible then to eval-

DD

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(2) pi.9-env^3f

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—LTD-

(4) pl.9-oriC

5'-l.TRc04-

(5) pl.9-envC

m-

(61 pl.9-nefC

-LH> O-

FIG. 1. Native and mutant 1.9 expression plasmids. (A)Genetic organization of 1.9, showing translated sequences inthe three forward-reading frames (open rectangles). (B) On-scale schematic representation of the native and mutant 1.9 ex-

pression plasmids: (0) pi.9-wt; (1) pi.9-wt containing de SV40ori; (2) complete deletion of the 3' LTR of pi.9-wt, BGH-ter-minator, and SV40 ori positioned 3' of the env gene; (3) par-tial deletion of the 3' LTR of pi.9-wt, BGH-terminator, andSV40 ori positioned 3' of the nefgene; (4), (5), and (6) Identicalto (1), (2), and (3) except that the incomplete 5'-LTR has beenreplaced by the full-length 5'-LTR. Q 1.9-wt proviral genome;ra BGH-terminator (BGH-term); H, SV40 ori. 5'-LTRinc,Incomplete 5'LTR; 5'-LTRc, complete 5'-LTR; 3'-LTRinc, in-complete 3'-LTR.

uate the infectivity of the viruses produced in the sensitive cell-free and coculture infectivity tests with CEMxl74 target cells.The cell-free infectivity test permits quantitative evaluation ofnative relative to mutated virus infectivity. The coculture in-fectivity test can detect mutant viruses that have a reduced in-fectivity, such as observed with vif~ particles that can effi-ciently spread infection only by cell-to-cell contact.3031 Acell-free infectivity test of the native and mutant 1.9 viruses us-

ing pigtail macaque peripheral blood mononuclear cells

SIVsmmPBj 14-1.9 NONINFECTIOUS PARTICLES 857

kD200»92»69»

46»

B env envC nef nefC wt ori oriCmwT s1 T s1 'L s"l s "L s 'T s^T T'T T1

|á.i«ilil«.itil-•- I i Î * • i S

30» <

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,gpl60

-gpl20

-pS5

-P27

FIG. 2. SDS-polyacrylamide gel electrophoresis analysis un-der reducing conditions of radioimmunoprecipitated cellularlysates and ultracentrifuged supernatants of transiently trans-fected COS-1 cells. L, Cellular lysates; S, ultracentrifuged su-

pernatants. Positions of gpl60, gpl20, p55, and p24 are as in-dicated. Molecular weights (MW) are indicated in kD. Lanesfor COS-1 cells transfected with B (negative control, Bluescriptplasmid), env (pl.9-env), envC (pl.9-envC), nef (pl.9-nef),nefC (pl.9-nefC), wt (pl.9-wt), ori (pl.9-ori), and oriC (pl.9-oriC).

[I]14

12

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x 8

2 6

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18-,16

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0 5 10 15 20 25Days Post-Infection

10 15 20 25 30Days Post-Infection

10 15 20Days Post-Infection

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16-_ 14-E& 12-

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frïTT*..,10 15 20

Days Post-Infection Days Post-Infection10 15 20

Days Post-Infection

FIG. 3. Evaluation of the infectivity of the native and mutant 1.9 viruses on CEMxl74 cells. (I) Viral infectivity was evalu-ated by monitoring RT activity293 in the supernatants of infected CEMxl74 cells over time in three different infectivity tests. (A)Viral replication in CEMxl74 cells: 107 CEMxl74 cells were transiently transfected with 5 pg of native and mutant 1.9 plas-mids by the DEAE-dextran procedure.2* (B) Cell-free infectivity test: 2 X 107 CEMxl74 cells were infected with 600 pi of cell-free supernatants containing equivalent amounts of native and mutant virus produced by transiently transfected COS-1 cells for1 hr at 37°C. Subsequently, the cells were washed and resuspended in medium. (C) Coculture infectivity test: 48 hr posttrans-fection of COS-1 cells transiently transfected with 15 pg of native and mutated 1.9 plasmids CEMxl74 cells were added to theculture in a 1:1 ratio. In all tests cells were split every 4 to 5 days. (II) For each infectivity test viral replication was measuredeither in the cell-free supernatants by measuring the presence of RT activity (D) and Gag antigen (Ag) by a specific SIV GagELISA29c (E) or in cellular lysates by Gag Ag ELISA (F). Only the results for the coculture infectivity test are shown in (D),(E), and (F). Identical results were found when the infectivity tests were followed for as long as 90 days (data not shown). Thenoninfectious phenotype of mutant 1.9-env and -envC viruses was reproducible and maintained when using high concentrationsof these viruses, obtained by ultracentrifugation of large amounts of culture supernatants from transfected COS-1 cells, even ininfectivity tests performed over a period of 90 days (data not shown). The following plasmids were used: (A) Bluescript was

used as a negative control; (O) pl.9-env; (•) pl.9-envC; (D) pl.9-nef; ( ) pl.9-nefC; (O) pl.9-wt; (A) pl.9-ori; (A) pl.9-oriC.

858 DESCHAMPS ET AL.

ooc

Bluescriptpi.9-envpl.9-envCpl.9-nefpl.9-nefCp 1.9-wtp 1.9-oripl.9-oriC

10 15 20Days Post-Infection

ZS 30

FIG. 4. Infectivity of the mutant 1.9 viruses on pigtailmacaque PBMCs as monitored by RT activity in cell-free cul-ture supernatants on the indicated days. PBMCs (5 X 105) pre-viously activated with concanavalin A (ConA) and grown inmedium containing recombinant human interleukin 2 (IL-2)were infected with 300 pi of virus (106 RT activity) obtainedfrom transfected COS-1 cells for 16 hr at 37°C. The cells were

subsequently washed and resuspended in culture medium.Viruses tested were produced by transient transfection ofCOS-1 cells with the plasmids indicated in the legend. Half theculture medium was changed every 3 to 4 days. The arrow in-dicates the day that freshly stimulated PBMCs were added tothe culture. Similar results were obtained when evaluating in-fectivity of the genetically altered 1.9 viruses transiently pro-duced by CEMxl74 cells (data not shown).

(PBMCs) as target cells was also performed to anticipate theinfectivity of the mutant virions in vivo (Fig. 4).

The viruses produced by the native plasmids pi.9-wt, pil-ori, and pl.9-oriC yielded a spreading infection with expectedsimilar infectivity kinetics and a peak of RT activity approxi-mately 9 days postinfection (Figs. 3 and 4). The infectivity pro-files of the mutant 1.9-nef and 1.9-nefC viruses were delayedin their peak of RT activity by approximately 7 days comparedwith the native particles (Figs. 3 and 4). However, the mutantviruses 1.9-nef and 1.9-nefC were as infectious as the nativeviruses because of their equivalent peak of RT activity. Thisdelay in kinetics did not appear to be a permanent feature ofthese viruses because they reverted to the native phenotype af-ter a second consecutive cell-free infection of CEMxl74 cells(Fig. 5). Preliminary PCR and sequence analysis of the nef-3'LTR region of several 1.9-nef and -nefC proviral sequencessuggested that a genetic event occurred in the in vitro culturethat reconstituted a replication-competent 3' LTR. The 1.9-nef3' LTR contained a mixed population of two viruses that hadeither a sequence indistinguishable from the wild type or a par-tially deleted 3' LTR sequence, where the 1.9-nefC 3' LTR hadreverted to the wild-type sequence. This observation suggestedthat rapid reconstitution of a replication-competent 3' LTR bygenetic recombination occurs when LTR sequences with a min-imal U3 overlap (161 bp for 1.9-nef and 408 bp for 1.9-nefC)are present at both ends of the proviral genome. Thus, to pro-duce fully inactivated virions, reconstitution of the genomethrough overlapping segments must be avoided.

Although the mutant viruses, 1.9-env and -envC, producedby the most genetically altered plasmids were identical in pro-tein content and maturation to the wild-type 1.9 virus, they were

unable to establish a productive infection in CEMxl74 cells andpigtail macaque PBMCs in all three infectivity tests (Figs. 3and 4). Thus, a comparison of the structure and infectivity ofthe four 1.9 mutant viruses produced after transfection led tothe identification of two fully inactivated 1.9 mutant viruses:1.9-env and -envC.

In an attempt to generate a substrate suitable for the pro-duction of large quantities of antigen for vaccination studies, a

stable Vero cell Une producing the noninfectious mutant 1.9-env particles, v/PBjl.9 env, was established by cotransfectionof pi.9-env and pSV2neo32 using the calcium phosphatemethod.33 The native plasmid p 1.9-ori was also used to estab-lish the cell line v/PBj 1.9 ori as a positive control. Both celllines were the result of selecting Geneticin-resistant clones andsubcloning. Similar to the study described above, analysis ofthe expression, maturation, and infectivity of the viruses pro-duced by these cell lines indicates that viral antigens are asso-

ciated with the virions in the culture supernatant and that the1.9-env viruses are not infectious in CEMxl74 cells and pig-tail macaque PBMCs (data not shown). Furthermore, the mor-

phogenesis of mutant 1.9-env viral particles produced by cellline v/PBjl.9 env was examined by thin-section electron mi-croscopy as described previously.34 The established cell linesproduced particles typical of the lentiviras family that did nothave any abnormalities in virus assembly or maturation (Fig.

oco

<t-

-&- Bluescript1.9-nef (CEMxl 74)1.9-nef (cocu)1.9-nefC (CEMxl 74)1.9-nefC (cocu)1.9-oriC (CEMxl74)

10 15 20 25Days Post-Infection

.-so 35

FIG. 5. Analysis of the infection kinetics of mutant viruses1.9-nef and -nefC after a second infection on CEMxl74 cells.Equal amounts of viruses 1.9-nef and -nefC, as measured byRT activity, obtained at the peak of infection of the transfectedCEMxl74 cells (Fig. 3A) and the coculture infectivity test (Fig.3C) were used to infect fresh CEMxl74 cells in a cell-free in-fectivity test. Similarly, an equal amount of 1.9-oriC virus, ob-tained from supernatants at the peak of infection in the cocul-ture infectivity test (Fig. 3C), was used as a positive control.Supernatants from CEMxl74 cells transfected with theBluescript plasmid was used as a negative control. Viral repli-cation was measured on the indicated days by measuring theRT activity found in cell-free culture supernatants. (CEMxl74),Virus obtained from transfected CEMxl74 cells; (cocu), virusobtained from the coculture infectivity test.

SIVsmmPBjl4-1.9 NONINFECTIOUS PARTICLES

[A] [B] a)

FIG. 6. Electron micrographs of thin section of Vero cell lines v/PBj 1.9 env and v/PBj 1.9 ori. [A] v/PBj 1.9 ori producing ma-ture infectious 1.9-ori viruses: (a) in vacuoles; (b) in the supernatant. [B] v/PBj 1.9 env producing 1.9-env mutant viruses: (a)mature particle in the supernatant; (b) budding virion with a fringe of surface knobs. Original magnification: X 120,000.

6). The majority of the particles were mature, typified by a cone-

shaped, condensed core and the occurrence of some sheddingof the envelope glycoproteins knobs.35 Immature and buddingstructures were only rarely observed. While most of the parti-cles were observed outside the cell at the plasma membrane, a

few mature structures were also observed in vacuoles, proba-bly after being ingested by phagocytosis.

The level of 1.9-env and -ori virus production by their re-

spective cell lines was estimated as 30 ng of p21gag per milli-liter of supernatant, based on the amount of p21gag protein de-tected in ultracentrifuged supernatants using a commercial SIVp27 ELISA kit (Coulter, Hialeah, FL). Although comparison ofthe expression levels of two independently established Vero celllines does not discriminate between subtle differences in ex-

pression level, the equivalent amount of 1.9-env and -ori virusesproduced by these cell lines suggested that the presence or ab-sence of nef in the absence of the NRE, does not dramaticallyinfluence in vitro virion production. The presence of SV40 ori

in all plasmids prevented further evaluation of the effect of Nefand the NRE on transcription in COS-1 cells. It is likely thatthe low production yield of the 1.9-env particles by cell linev/PBj 1.9-env could be improved by replacing the 5' LTR byconstitutive strong or inducible heterogeneous promoters.

The structurally intact genetically inactivated particles we

constructed and characterized have two advantages over wholeinactivated particles: (1) the structure of the genetically inacti-vated particles remains intact, which means that all potentialimmunogenic determinants are present in their native form; and(2) the genetically inactivated particles are potentially safer thanwhole inactivated particles. Several other groups have demon-strated the feasibility of producing noninfectious HIV and SIVvirus-like particles,36-40 including S. A. Gonzalez et al, whoproduced recombinant vaccinia 1.9 virus-like particles con-

taining the matrix protein and the Env proteins.41 Although thescope of our work was to design a model immunogen for theSIV-monkey system with the minimal deletions necessary for

Xf o DESCHAMPS ET AL.

complete inactivation, the safety of the 1.9-env and -envC mu-tant virions or mutant proviral DNA vaccine would have to beimproved by introducing additional mutations/deletions in theproviral genome prior to their use for human immunization(e.g., deletion of RNA encapsidation site, inactivation of viralprotease and RT).

We have defined the minimal conditions required for the ge-netic construction of noninfectious 1.9 proviral DNA, and char-acterized the fully assembled, noninfectious 1.9-env and envCmutant viral particles produced in vitro. Because the Vero celllines did not produce sufficient virus for vaccine evaluation, we

decided to determine the immunogen and vaccine potential ofthe 1.9-env particles using DNA immunization. DNA immu-nization has several advantages over virus particle immuniza-tion: (1) the vaccine preparation is easy and inexpensive; (2)regulatory and accessory genes, not present in the virion, are

expressed in vivo and can stimulate an immune response; and(3) DNA immunization has been shown to be particularly suit-able for inducing a cytotoxic T lymphocyte immune response,whose role in the protection against HIV infection has beenshown to be important.42-44 Ongoing DNA immunization andvaccination experiments will determine whether the mutant viri-ons produced on injection of the genetically altered 1.9-env and-envC proviral DNA in monkeys are both noninfectious in vivoand capable of inducing protective humoral and cellular im-mune responses. These experiments should also provide insightinto the role of specific viral antigens in the induction of a spe-cific cellular or humoral immune response.

ACKNOWLEDGMENTS

We thank C. Vanhulle and R. Legas for their expert techni-cal assistance. We thank SmithKline Beecham Biologicals forallowing us to use their facilities to work with the wild-typeSIVsmmPBj 14-1.9 virus. We are grateful to D. Labbe for teach-ing us to work with SIV in a P3 facility. We thank S. Dewhurstfor providing the molecular clone SIVsmmPBj 14-1.9, P. Fultzfor the anti-SIV serum, and C. Thiriart for the anti-SIV poly-clonal and monoclonal antibodies. We also thank K. Willard-Gallo for critical reading of the manuscript. This research was

supported by a grant of the Fonds National de la RechercheScientifique and Grant AI27136 of the NIAID, NIH. M.D. issupported by a fellowship of the Institut pour l'Encouragementde la Recherche Scientifique dans l'Industrie et l'Agricultureand the Caisse Générale d'Epargne et de Retraite.

REFERENCES

1. Contag CH, Dewhurst S, Viglianti GA, and Mullins H: Simian im-munodeficiency virus. In: The Human Retroviruses (Gallo RC andlay G, eds.). Academic Press, San Diego, California, 1991, pp.245-276.

2. Zhang I, Martin LN, Watson EA, Montelaro RC, West M, EpsteinL, and Murphy-Corb M: Simian immunodeficiency virus/Delta-in-duced immunodeficiency disease in rhesus monkeys: Relation ofantibody response and antigenemia. J Infect Dis 1988; 158:1277-1286.

3. Kestler HW III, Kodama T, Ringler D, Marthas M, Pedersen N,

Lackner A, Regier D, Seghal P, Daniel M, King N, and DesrosiersR: Induction of AIDS in rhesus monkeys by molecularly clonedsimian immunodeficiency virus. Science 1990;248:1109-1112.

4. Fultz PN, McClure HM, Anderson DC, Swenson RB, Anand R,and Srinivasan A: Isolation of a T-lymphotropic retrovirus fromnaturally infected sooty mangabey monkeys (Cercocebus atys).Proc Nati Acad Sei USA 1986;83:5286-5290.

5. Fultz PN, McClure HM, Anderson DC, and Switzer WM:Identification and biologic characterization of an acutely lethalvariant of simian immunodeficiency virus from sooty mangabeys(SIV/SMM). AIDS Res Hum Retroviruses 1989;5:397^I09.

6. Martin MA: SIV pathogenicity. Fast acting slow viruses. Nature(London) 1990;345:572-573.

7. Fultz PN and Zack PM: Unique lentivirus-host interactions:SIVsmmPBjl4 infection of macaques. Virus Res 1994;32:205-225.

8. Dewhurst S, Embretson IE, Anderson DC, Mullins II, and FultzPN: Sequence analysis and acute pathogenicity of molecularlycloned SIVsmmPBj 14. Nature (London) 1990;345:636-640.

9. Israel ZR, Dean GA, Maul DH, O'Neil SP, Dreitz MI, Mullins II,Fultz PN, and Hoover EA: Early pathogenesis of disease causedby SIVsmmPBj 14 molecular clone 1.9 in macaques. AIDS ResHum Retroviruses 1993;9:277-286.

10. Hu WS and Temin HM: Retroviral recombination and reverse tran-

scription. Science 1990;250:1227-1233.11. Gilboa E, Mitra S, Goff S, and Baltimore D: A detailed model of

reverse transcription and tests of crucial aspects. Cell 1979; 18:93-100.

12. Varmus H and P. Brown: Retroviruses. In: Mobile DNA (Berg DEand Howe MM, eds.). American Society for Microbiology Press,Washington, D.C., 1989, pp. 53-108.

13. Kestler HW III, Ringler DI, Mori K, Panicali DL, Seghal PK,Daniel MD, and Desrosiers RC: Importance of the nef gene formaintenance of high virus load and for the development of AIDS.Cell 1991;65:651-662.

14. Daniel MD, Kirchhoff F, Czajak SC, Seghal PK, and DesrosiersRC: Protective effects of a live attenuated SIV vaccine with a dele-tion in the nef gene. Science 1992;258:1938-1941.

15. Antoni BA, Stein SB, and Rabson AB: Regulation of human im-munodeficiency virus infection: Implications for pathogenesis. AdvVirus Res 1994;43:53-145.

16. Lu Y, Stenzel M, Sodroski IG, and Haseltine WA: Effects of longterminal repeat mutations on human immunodeficiency virus type1 replication. I Virol 1989;63:4115^1119.

17. Guy B, Acres RB, Kieny MP, and Lecocq IP: DNA binding fac-tors that bind to the negative regulatory element of the human im-munodeficiency virus-1 : Regulation by nef. 3 Acquir Immune DeficSyndr 1990;3:797-809.

18. Jones KA, Luciw PA, and Duchange N: Structural arrangementsof transcriptional control domains within the 5' untranslated re-

gions of the HIV-1 and HIV-2 promoters. Genes Dev 1988;2:1101-1114.

19. Gaynor R: Cellular transcription factors involved in the regulationof HIV-1 gene expression. AIDS 1992;6:347-363.

20. Niederman TMJ, Thielan BJ, and Ratner L: Human immunodefi-ciency virus type 1 negative factor is a transcriptional silencer. ProcNati Acad Sei USA 1989;86:1128-1132.

21. Niederman TMJ, Hu W, and Ratner L: Simian immunodeficiencyvirus negative factor suppresses the level of viral mRNA in COScells. J Virol 1991;65:3538-3546.

22. Miller MD, Warmerdam MT, Gaston I, Greene WC, and FeinbergMB: The human immunodeficiency virus-1 nef gene product: Apositive factor for viral infection and replication in primary lym-phocytes and macrophages. I Exp Med 1994;179:101-113.

23. Hammes SR, Dixon EP, Malim MH, Cullen BR, and Greene WC:

SIVsmmPBjl4-1.9 NONINFECTIOUS PARTICLES 861

Nef protein of human immunodeficiency virus type 1 : Evidenceagainst its role as a transcriptional inhibitor. Proc Nati Acad SeiUSA 1989;86:9549-9553.

24. Woychik RP, Lyons RH, Post L, and Rottman FM: Requirementfor the 3' flanking region of the bovine growth hormone gene foraccurate polyadenylylation. Proc Nati Acad Sei USA 1984;81:13944-3948.

25. Pfarr DS, Rieser LA, Woychik RP, Rottman FM, Rosenberg M,and Reff ME: Differential effects of polyadenylation regions on

gene expression in mammalian cells. DNA 1986;5:115-122.26. Cullen BR: Regulation of human immunodeficiency virus replica-

tion. Annu Rev Microbiol 1991;45:219-250.27. Vaishnav YN and Wong-Staal F: The biochemistry of AIDS. Annu

Rev Biochem 1991;60:577-630.28. Chittenden T, Frey A, and Levine AJ: Regulated replication of an

episomal simian virus 40 origin plasmid in COS7 cells. J Virol1991;65:5944-5951.

29. Salter RD, Howell DN, and Cresswell P: Genes regulating HLAclass I antigen expression in T-B lymphoblast hybrids. Immuno-genetics 1985;21:235-246.

29a. Willey RL, Smith DH, Lasky LA, Theodore TS, Earl PL, MossB, Capon DJ, and Martin MA: In vitro mutagenesis identifies a re-

gion within the envelope gene of the human immunodeficiencyvirus that is critical for infectivity. J Virol 1988;62:139-147.

29b. Dorsett DL, liana K, and Winocour E: Quantitation of a simianvirus 40 nonhomologous recombination pathway. J Virol1983;48:218-228.

29c. Thiriart C, Francotte M, Cohen J, Collignon C, Delers A,Kümmert S, Molitor C, Gilles D, Roelants P, Van Wijnendaele F,De Wilde M, and Brück C: Several antigenic determinants exposedon the gpl20 moiety of HIV-1 gpl60 are hidden on the mature

gpl20. J Immunol 1989;143:1832-1836.30. Strebel K, Daugherty D, Clouse K, Cohen D, Folks T, and Martin

MA: The HIV "A" (sor) gene product is essential for virus infec-tivity. Nature (London) 1987;328:728-730.

31. Fan L and Peden K: Cell-free transmission of Vif mutants of HIV-1. Virology 1992;190:19-29.

32. Mulligan RC and Berg P: Expression of a bacterial gene in mam-

malian cells. Science 1980;209:1422-1427.33. Wigler M, Pellicer A, Silverstein S, Axel R, Urlaub G, and Chasin

L: DNA-mediated transfer of the adenine phosphoribosyltrans-ferase locus into mammalian cells. Proc Nati Acad Sei USA1979;76:1373-1376.

34. Gelderblom HR, Hausmann EHS, Özel M, Pauli G, and Koch MA:Fine structure of human immunodeficiency virus (HIV) and im-munolocalization of structural proteins. Virology 1987;156:171-176.

35. Gelderblom HR: Assembly and morphology of HIV: Potential ef-fect of structure on viral function. AIDS 1991;5:617-637.

36. Gheysen D, Jacobs E, de Foresta F, Thiriart C, Francotte M, ThinesD, and de Wilde M: Assembly and release of HIV-1 precursorpr55ias virus-like particles from recombinant baculovirus-infectedcells. Cell 1989;59:103-112.

37. Haffar O, Garrigues J, Travis B, Moran P, Zarling J, and Hu SL:Human immunodeficiency virus-like, nonreplicating, gag-env par-ticles assemble in a recombinant vaccinia virus expression system.J Virol 1990;64:2653-2659.

38. Haynes JR, Cao SX, Rovinski B, Sia C, James O, Dekaban GA,and Klein M: Production of immunogenic HIV-1 viruslike parti-cles in stably engineered monkey cell lines. AIDS Res HumRetroviruses 1991;7:17-27.

39. Rovinski B, Haynes JR, Cao SX, James O, SIA C, Zolla-Pazner S,Matthews TJ, and Klein MH: Expression and characterization ofgenetically engineered human immunodeficiency virus-like parti-cles containing modified envelope glycoproteins: Implications fordevelopment of a cross-protective AIDS vaccine. J Virol1992;66:4003^1012.

40. Kräusslich HG, Ochenbauer C, Traenckner AM, Mergener K,Fäcke M, Gelderblom HR, and Bosch V: Analysis of protein ex-

pression and virus-like particle formation in mammalian cell linesstably expressing HIV-1 gag and env gene products with or with-out active HIV proteinase. Virology 1993;192:605-617.

41. Gonzalez SA, Affranchino JS, Gelderblom HR, and Burny A:Assembly of the matrix protein of simmian immunodeficiencyvirus into virus-like particles. Virology 1993;194:548-556.

42. Wang B, Boyer J, Srikantan V, Coney L, Carrano R, Phan C, MervaM, Dang K, Agadjanan M, Gilbert L, Ugen KE, Williams WV, andWeiner DB: DNA inoculation induces neutralizing immune re-

sponses against human immunodeficiency virus type 1 in mice andnonhuman primates. DNA Cell Biol 1993;12:799-805.

43. Ulmer JB, Donnelly JJ, Parker SE-, Rhodes GH, Feigner PL, DwarkiVJ, Gromkowski SH, Deck RR, DeWitt CM, Friedman A, HaweLA, Leander KR, Martinez D, Perry HC, Shiver JW, MontgomeryDL, and Liut MA: Heterologous protection against influenza by in-jection of DNA encoding a viral protein. Science 1993;259:1745-1749.

44. Levy JA: Pathogenesis of human immunodeficiency virus infec-tion. Microbiol Rev 1993;57:183-289.

Address reprints requests to:Marguerite Deschamps

ULBDepartment of Molecular Biology

Laboratory of Biological Chemistry/Prof. A. Burnyrue des chevaux, 67

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