Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4114-4118, May 1992 Microbiology Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity T. NAGASHUNMUGAM*, A. VELPANDI*, C. S. GOLDSMITHt, S. R. ZAKIt, V. S. KALYANARAMANt, AND A. SRINIVASAN*§ *The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104; tDivision of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333; and tAdvanced Bioscience Laboratories, Inc., 5510 Nicholson Lane, Kensington, MD 20805 Communicated by Hilary Koprowski, January 16, 1992 ABSTRACT In an effort to understand the contribution of retroviruses make use of tryptophan tRNA. Sequence anal- the primer-binding site (PBS) region to human immunodefi- ysis of HIV-1 revealed PBS corresponding to the lysine ciency virus (HIV) replication, we have constructed a mutant tRNA as in other lentiviruses (7). It has also been recently HIV proviral DNA with an alteration in the 5' end of the PBS. demonstrated that HIV RT forms a stable complex with The PBS mutant proviral DNA was characterized by transfec- isoacceptor 3 of lysine tRNA (Lys3 tRNA) (8), as has been tion of the viral DNA into CD4' and non-CD4' target cells. demonstrated for the RT enzyme of avian viruses. The results indicate that mutation in the PBS reduced the level Initiation of viral DNA synthesis is a crucial step in the of viral particles released into the medium of transfected cells replication of retroviruses, and the essential PBS is embed- in comparison to wild-type proviral DNA. The viral particles ded in the complex structure ofRNA (9). A number ofstudies were noninfectious upon transmission to established CD4' cell have reported the importance of the secondary structure of lines and phytohemagglutinin-stimulated peripheral blood the genome at the 5' end in the assembly and infectivity ofthe lymphocytes. Electron microscopic analysis of the transfected virus (10-12). In an effort to understand the structural cells revealed no abnormalities in the structure of the virion requirement of the PBS in reverse transcription and virus directed by the mutant proviral DNA. Also, the protein and morphogenesis, we have introduced an insertional mutation RNA contents of the mutant virions were similar to the wild in the PBS of HIV-1. The results obtained with the PBS type. The quantitation of intracellular viral structural protein mutant imply that the PBS region, in addition to its partici- in the transfected cells, however, indicated that the PBS pation in reverse transcription, is also involved in the assem- mutation may have an effect on the assembly of viral particles bly of viral particles. in addition to completely abolishing reverse transcription of viral RNA into DNA. These results provide evidence that the MATERIALS AND METHODS PBS region of the viral genome has multiple functions in HIV-I Cell Lines. A human rhabdomyosarcoma (RD) cell line replication. (American Type Culture Collection) was maintained as a monolayer culture in Dulbecco's modified Eagle's medium The genome of human immunodeficiency virus type 1 (HIV- supplemented with 10% fetal bovine serum, penicillin (100 1), like other members of the lentivirus family, encodes not units/ml), and L-glutamine (540 pug/ml) at 370C with 5% CO2. only the virion proteins gag, pol, and env common to all CEM x 174 and HUT78 cells were maintained as suspension replication-competent retroviruses but also eight accessory cultures in RPMI 1640 medium; phytohemagglutinin- genes, some of which are involved in the gene regulation and stimulated (10 Ag/ml) peripheral blood lymphocytes were morphogenesis of HIV (1-3). Biochemical and structural grown in RPMI 1640 medium containing T-cell growth factor studies have revealed that HIV is about 80-120 nm in (10%). diameter and consists of an inner core (nucleoid) surrounded Construction of the PBS Mutant. The HIV proviral DNA by an outer envelope. The core corresponds to a ribonucleo- plasmid designated pARV was derived from cells infected protein surrounded by the capsid protein. The viral RNA with HIVSF2 (13). The pARV plasmid was cleaved with the genome is in the ribonucleoprotein, and it is composed oftwo restriction enzyme Nar I, and the resultant linear molecule identical positive-sense RNAs with a 5' cap and a 3' poly(A) was purified by agarose gel electrophoresis. The Nar I structure (4). overhang sequence in the DNA was filled in by using Klenow The replication cycle of HIV-1 and other retroviruses polymerase and ligated by using T4 ligase (14). The recom- involves the reverse transcription of virion RNA to viral binant plasmid obtained from the transformed colonies was DNA, which then becomes incorporated into the host cellular checked for the presence of the Nar I cleavage site. The genome. The reverse transcription is mediated by the virion- plasmid (pARV-NAR) lacking the Nar I site was further associated enzyme reverse transcriptase (RT) (5, 6). As with confirmed by sequence analysis with a Sequenase kit (United all DNA polymerases, RT needs a primer covering a 3' States Biochemical). hydroxyl (OH) group to initiate cDNA synthesis. The in vivo Transfection. RD cells (1 x 106 cells per plate) were split 24 primer for the viral first-strand cDNA synthesis from an RNA hr before transfection, and growth medium was replaced 1-2 template has been shown to be tRNA (6). A region near the hr before the addition of calcium phosphate-precipitated 5' end of the viral RNA genome designated as the primer- DNA (15). Cells were exposed to the precipitate for 8 hr binding site (PBS) is complementary to the 18 nucleotides of followed by a 90-sec glycerol shock (16). For DEAE-dextran the 3' CCA end ofthe specific tRNA primer. Predominantly, transfection, CEM x 174 cells (1 x 107) were washed and many mammalian retroviruses use proline tRNA, and avian Abbreviations: RT, reverse transcriptase; PBS, primer-binding site; The publication costs of this article were defrayed in part by page charge RD, rhabdomyosarcoma; HIV-1, human immunodeficiency virus payment. This article must therefore be hereby marked "advertisement" type 1; RIPA, radioimmunoprecipitation analysis. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 4114 Downloaded by guest on September 24, 2021 Microbiology: Nagashunmugam et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4115 PBS the PBS was confirmed by restriction enzyme and sequence LTR GAG POt ENV LEF analyses. Biological Characterization of HIV-1 Proviral DNA with an Altered PBS. To test the mutant for replication competence, TGGCGCCCGAACAGGGAC pARV and mutant proviral DNAs were directly trans- Nar I wild-type fected into a CD4' cell line designated CEM X 174. A TGGCGcgCCCGAACAGGGAC pARV-NJAR positive result is indicative of a spreading HIV-1 infection in this CD4' cell line. Transfected CEM x 174 cells with FIG. 1. Structure of wild-type and Imutated proviral DNAs used wild-type proviral DNA showed high levels of HIV-1 p24 in this study. The wild-type PBS seque nce is shown. The insertion antigen in the medium, in comparison to the PBS mutant mutation in the PBS region of the muitant proviral DNA (pARV- (Table 1). These results indicate that virus particles directed NAR) is indicated in lowercase letters. LTR, long terminal repeat. by the mutant proviral DNA are unable to initiate a spreading infection. .iumcontainin100 of suspended in 1 ml of serum-free medium containing 100,.ag of Effect of the PBS Mutation on Virus Production. To distin- DEAE-dextran and 10 ,ug ofplasmi( iDNA for 1-2 hr inaPetri guish the effect of the mutation production (synthesis dish. The cells were washed once and were grown in RPMI with 109woserum (17). and assembly of viral particles), we have carried out trans- fection experiments using a transient expression system assay (Coul- HIV Antigen and RT Assay. The HIV antigen(17).en assay (Coul- involving monolayer cells. Human RD cells ideal for ter) was performed according to t these studies, because they have been shown to release high lines to quantitate the amount of herusmanufacTer'assguid procedure was essentially similar to oruat(d8)criTbedR(T asThe levels of viral particles upon transfection as monitored by RT oi and a viral antigen assay (24-26). Transfection of the mutant endogenous RT reaction was carrierd outthatdescribsedn(9In the absence ofofthethe proviral DNA consistently showed a 40-60% reduction in the exogenous template poly(A)-(dT)12 Immunoprecipitation Analysis. i 1D cells102)(2 x trans- release of viral particles in comparison to wild type. The fected with 10 of or 10 IDgof mutant pARV-NAR differences observed in the extracellular viral p24 prompted jig pARV , us agtof mutantol Th to quantitate the intracellular p24 in cells transfected with proviral DNA were labeled 60 hr after glycerol shock. The mutant and wild-type proviral DNA (Table 2). Unlike extra- labeling continued for 12 hr with 8 ml of methionine- and cellular p24 levels, intracellular p24 levels similar in cysteine-deficient serum-free mediu m containinge50r Ciu( Ci both wild-type and mutant proviral DNA-transfected cells = 37 GBq) of Tran35S-label per ml. The cell-free medium and (Table 3). (al ) cells were analyzed by using HIV--positive patient sera and The viral particles released from the transfected cultures normal human sera (21). werre fixed for EM with 2.5% infectivity potential. Since our previous EM. After transfection, cells emixm edtfordEM studies (27) and others (28) have shown that the proviral clone glutaraldehyde, postfixed with osi tetroxide, and em- pARV-derived virus is not able to infect CEM 174 cells bedded in Sectionisum Epon/Araldite. a cell-free virus, we have used the cocultivation method to citrate and uranyl acetate (22).