VIROLOGY

175,139-l 48 (1990)

Production of Human lmmunodeficiency Virus (HIV)-like Particles from Cells Infected with Recombinant Vaccinia Viruses Carrying the gag Gene of HIV TATSUO SHIODA’ Department

AND

HIROSHI SHIBUTA’

of Viral Infection, Institute of Medical Science, University of Tokyo, Minato-ku,

Tokyo 108, Japan

Received June 2, 1989; accepted October 30, 1989

We constructed a recombinant vaccinia virus carrying the entire gag and pal genes of human immunodeficiency virus type 1 (HIV-l). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of loo-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pal proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pal gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production. o 1990 Academic

Press. Inc.

vaccinia vector contains the HIV protease gene following the gag genes (Walker et a/., 1987; Flexner et a/., 1988; Gowda et al., 1989). The primary translational product of the HIV gag gene is a polyprotein precursor of 55K Da (~5% which is subsequently processed into the mature ~17, ~24, and pl5 gag proteins (Mervis et a/., 1988; Veronese et a/., 1988), a process that is mediated by the HIV protease encoded in the subsequent pal gene (Kramer et a/., 1986; Madisen et a/., 1987; Debouck et al., 1987; Kr$iusslich er a/., 1988). The gag and PO/genes are not in frame and are partially overlapping (Muesing et a/., 1985; Ratner et a/., 1985; Sanchez-Pescador et a/., 1985; Wain-Hobson et a/., 1985), but PO/ gene products are expressed initially as a gag--PO/ fusion polyprotein which is processed into the protease, reverse transcriptase (RT), integrase, and mature gag proteins. The gag-polfusion protein is generated by a ribosomal frame shifting event at the specific site located in the overlapping region of the gag and PO/ open reading frames (Jacks et a/., 1988; Wilson et a/., 1988). We also constructed a recombinant vaccinia virus which carries the gag and PO/ genes of HIV and found that HIV-like particles containing both the active RT and the processed gag proteins were produced from SW480 cells infected with this recombinant virus, although the main gene product detected within the cells was the gag precursor protein. MATERIALS AND METHODS Cells Green Monkey kidney CV-1 cells, human embryonal lung R66 cells, and HeLa cells were grown in Eagle’s

INTRODUCTION Recently, increasing attention has been paid to the gag antigens of human immunodeficiency virus (HIV). Specific decreases in the level of anti-gag antibodies have been noticed in patients before the development of severe clinical symptoms of acquired immunodeficiency syndrome (AIDS) (Schtipbach et al., 1985; Lange et al., 1986; Weber et a/., 1987). A monoclonal antibody against ~17, one of the mature gag proteins, has been repot-ted to possess a strong neutralizing activity against HIV (Papsidero ef a/., 1989). Furthermore, cellular immunity against viral core proteins is now known to play a definitive role in recovery from viral infections (Townsend et a/., 1984; Yewdell er a/., 1985; McMichael ef a/., 1986), and cellular immunity against gag proteins has been detected in HIV infections (Plata et al., 1987; Wahren et al., 1987; Walker et al., 1987; Nixon eTa/., 1988; Reddy and Grieco, 1989). Several workers have tried to express HIV gag proteins in insect cells by using a baculovirus vector (Madisen et a/., 1987), in yeast cells by using a plasmid vector (Kramer et a/., 1986), and in mammalian cells by using a recombinant vaccinia vector (Walker et a/., 1987; Flexner et a/., 1988; Koup et a/., 1989; Gowda et a/., 1989). However, although the gag precursor is processed in insect cells and yeast cells, it is not efficiently processed in mammalian cells even when the ’ Present address: % Dr. J. A. Levy, University of California, School of Medicine, Cancer Research Institute, M-l 282, San Francisco, CA 94143-0128. ‘To whom requests for reprints should be addressed. 139

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Copynght 0 1990 by Academic Press. Inc. All rights ot reproduction I” any tOrm r~?servea.

SHIODA AND SHIBUTA

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FIG. 1. Diagram showing cDNA regions of HIV inserted into the vaccinia genome. The cDNA was prepared from an infectious cDNA clone, pNL4-3-2, by digestion with the indicated endonucleases, inserted into the vaccinia vector plasmid pNZ68K2, and subsequently transferred to vaccinia WR virus by homologous recombination.

minimum essential medium (MEM) containing 5% newborn calf serum. Human colon carcinoma SW480 cells (Adachi et a/., 1986) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS). The human lymphoid cell lines MT-4, Molt-4, and HPB-ALL were grown in RPMI 1640 medium supplemented with 10% FCS. Plasmid DNA An infectious molecular DNA clone of HIV type 1, pNL4-3-2 (Adachi eta/., 1986; Strebel eta/., 1987), was kindly supplied by Dr. A. Adachi, Virus Research Institute at Kyoto University. The pNL4-3-2 and vaccinia vector plasmid pNZ68K2 (Gotoh et al., 1989) were propagated in Escherichia co/i strain DH 1. Construction of the recombinant plasmids DNA fragments of pNL4-3-2 were inserted into the multicloning site of pNZ68K2 as shown in Fig. 1. A DNA fragment containing the entire coding region of the HIV gag and pal genes was isolated by cutting pNL4-3-2 at the Bg/ll site (nucleotide position 226 from the mRNA starting site) and the EcoRl site (nucleotide position 5293) and was inserted into the plasmid between the BarnHI and EcoRl sites, The resultant plasmid was designated as pNZ-gag/pa/. To align thegagandpolgenes in the same open reading frame, the Sg/ll restriction site of pNL4-3-2 (nucleotide position 1643) was cut, repaired by Klenow polymerase, and then religated. This procedure inserted 4 base pairs (GATC) at the overlapping region of the gag and pol open reading

frames, replacing the carboxy-terminal 63 amino acid residues of the 15 K gag protein with the complete pal protein. The normal ribosomal frame shifting site is located 6 bases upstream of this Bglll site (Jacks et a/., 1988), and the translational product of this mutated gene had an amino acid sequence of glycine-lysineisoleucine instead of the arginine-glutamic acid at amino acid positions 433 and 434 of the natural gagpal fusion protein. The gag-pol region thus mutated was then introduced in the pNZ68K2 plasmid, yielding pNZ-gag-pal-fuse. To construct pNZ-gag/pro, which carried the entire gag gene and the protease coding region but lacked the 3’ proximal half of the pal gene, a 2.6-kilobase (kb) fragment prepared by cutting pNL4-32 at the Bglll site (nucleotide position 226) and the Pvull site (nucleotide position 2847) was inserted into the pNZ68K2 plasmid between the BarnHI and Smal sites. To construct pNZ-gag, which carried the gag gene alone, a 1.8-kb fragment prepared by cutting the pNL43-2 at the Bglll site (nucleotide position 226) and the Hincll site (nucleotide position 2044) was inserted in the pNZ68K2 plasmid between the BarnHI and Smal sites. These plasmids were all propagated in E. co/i strain DH 1. Construction of the recombinant vaccinia viruses Transfer of the HIV genes from the recombinant plasmids, pNZ-gag/pa/, pNZ-gag-pal-fuse, pNZ-gag/pro, and pNZ-gag, to vaccinia virus WR was achieved by the standard homologous recombination method using CV-1 cells (Mackett et al., 1985; Gotoh et al., 1989).

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The recombinant vaccinia viruses thus obtained were designated as Vat-gag/pal, Vat-gag-pal-fuse, Vacgag/pro, and Vat-gag, and were propagated in CV-1 cells.

a commercial kit for the detection of HIV-1 antigens (ELAVIA Agl).

lmmunoblot analysis

Samples were adsorbed onto a carbon grid, fixed with 2.5% glutaraldehyde in 0.1 ll/I carbohydrate buffer (pH 7.5) and then negatively stained with 2% uranyl acetate (pH 4.5). Samples were then viewed using a Hitachi-600 electron microscope.

CV-1 or SW480 cells in 35-mm culture plates were infected with WR or recombinant viruses at 20 plaque forming units (PFU) per cell and incubated at 37” for 24 hr in serum-free culture medium containing 40 pg per milliliter of cytosine arabinoside (Ara-C), which selectively inhibits the synthesis of vaccinia virus late proteins: Ara-C inhibits the growth of vaccinia virus, but allows the expression of 7.5 K promoter-driven foreign genes (Cochran et al., 1985). The cells were lysed in 200 ~1 of 1% sodium dodecyl sulfate (SDS)-1 25 mM Tris-HCI (pH 8.0) and boiled for 1 min. The cell lysates were adjusted to contain final concentrations of 10% glycerol, 5% 2-mercaptoethanol, and 0.04Obbromphenol blue. Then 20-~1samples were electrophoresed on SDS-l 0% polyacrylamide gel (SDS-PAGE) (Laemmli, 1970). Polypeptides resolved were electrically transferred to nitrocellulose filters (Towbin et a/., 1979). These were then incubated with serum from AIDS patients, a mouse monoclonal ascitic antibody, Vak-5, against the HIV gag p24 protein (Hattori et a/., 1987) or serum from mice immunized with the WR virus (Gotoh et al., 1989). The sera and ascitic fluid were used at dilutions of 1:50 to 1:lOO. Immobilized antibodies were detected using 1*51-labeledprotein A (30 mCi/mg) and autoradiography. The VAK-5 was kindly supplied by Dr. K. Sagawa of Kurume University. Northern blot hybridization Total RNA fractions were prepared from WR- or Vat-gag/PO/-infected SW480 cells by the guanidium thiocyanate method (Chirgwin et al., 1979). The RNA was resolved in 1% agarose gel containing 16% formaldehyde (Lehrach et a/., 1977) and was transferred to nitrocellulose filters (Thomas eta/., 1980) for hybridization with 32P-labeled HIV DNA probes. Reverse transcriptase (RT) assay The RT activity in culture fluids or in infected cells was assayed as described previously (Willey et a/., 1988) using poly(A) as the template and oligo(dT) as the primer. To prepare cytoplasmic fractions, infected cells were suspended in phosphate-buffered saline, freeze-thawed three times, and centrifuged at 3000 rpm for 10 min to remove nuclear debris. Estimation of the amount of HIV antigens The amount of HIV antigens was semiquantitatively estimated by a sandwich enzyme immunoassay using

Electron microscopy

Enzymes and other materials Ara-C was purchased from Sigma (St. Louis, MO); the multiprime DNA labeling system kits and all the radioactive compounds were from Amersham International Plc (Amersham, U.K.); bovine alkaline phosphatase, T4 DNA ligase, and all the restriction endonucleases were from Takara Shuzo (Kyoto, Japan); poly(rA) and oligo(dT),Z-,e were from Pharmacia (Piscataway, NJ); ELAVIA Agl kit was from Diagnostics Pasteur (Marnes La Coquette, France). RESULTS Expression of the gag and pal genes in CV-1 cells Expression of the gag protein in CV-1 cells from the Vat-gag/PO/, which contained the entire gag and pal genes, was examined by immunoblot analysis of the cell lysates. Infected cells were incubated at 37” for 24 hr in the presence of Ara-C. The result showed that the main polypeptide was detected at the position of the p55 gag precursor (Fig. 2). Signals at the position of the mature gag protein, ~24, were scarcely detected even after autoradiography with a long period of exposure. No RT activity was detected in the infected cells. However, the Northern blot analysis of RNAs prepared from the infected cells demonstrated that the HIV gene region inserted in the vaccinia viral genome was transcribed into mRNA of 5.5 kb long, which corresponded to the entire gag and pal gene region (data not shown). This observation suggested that a ribosomal frame shift producing the gag-pol fusion protein was rare in this experimental system. Therefore, we tried to express the gag-pol fusion protein by constructing another recombinant vaccinia virus, Vat-gag-PO/-fuse. lmmunoblot analysis of infected CV-1 cell lysates revealed that polypeptides of about 120K and 32K were synthesized (Fig. 2). Few signals were detected at the positions of the mature gag proteins. The 32K polypeptide seemed to correspond to the HIV integrase, which was located at the carboxyterminal portion of the gag-pal fusion protein. The 120K polypeptide seemed to correspond to the gagpal fusion protein lacking the carboxy-terminal inte-

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FIG. 2. lmmunoblot analysis of gene products. The lysates of CV1 cells infected with recombinant vaccinia viruses were electrophoresed on SDS-polyacrylamide gel and polypeptides were transferred to a nitrocellulose filter which was incubated with the serum of an AIDS patient. Immobilized antibodies were probed with radioactive protein A. Mock-infected and WR-infected CV-1 cells, pNL4-3-2transfected Molt-4 cells, and mock-infected Molt-4 cells were included as controls.

grase. This result indicated that in CV-1 cells the Vacgag-co/-fuse produced gag-pal fusion protein from which only the integrase was processed.

AND SHIBUTA

from infected SW480 cells, indicating that the lack of RT activity in CV-1 cell cultures was not caused by an inhibitor of the enzyme (data not shown). We also detected RT activity in the culture fluid of SW480 cells infected with Vat-gag/PO/. No RT activity was detected in all the cells infected with wild-type WR virus. The extracellular RT activity in SW480 cell cultures infected with Vat-gag-PO/-fuse was two- to fivefold higher than in those infected with Vat-gag/pa/. Since the retroviral gag and pal gene products are supposed to be nonsecretory proteins, we thought at first that the release of RT activity from the cells was caused by cell lysis from vaccinia virus infection. This may be the case for Vat-gag-PO/-fuse, since the RT activity in the cytoplasmic fraction was much higher than in the culture fluid from 12 to 36 hr postinfection (Fig. 4, right), suggesting that the RT activity in the culture fluid had leaked from dead cells. In Vat-gag/PO/ infection, however, the RT activity in cytoplasmic fraction showed a peak at 12 hr postinfection and decreased rapidly thereafter, while the activity in the culture fluid was still high 48 hr postinfection (Fig. 4, left). This result suggested that the RT molecules produced in Vat-gag/PO/-infected SW480 cells were rapidly released into the culture fluid before cell death. Release of HIV-like particles from SW480 cells infected with Vat-gag/pal In order to examine whether the RT activity released from Vat-gag/PO/-infected SW480 cells was in a solu-

RT activities in culture fluids of cells infected with recombinant vaccinia viruses Although there have been no reports suggesting the existence of a cellular factor concerned with the processing of the retroviral gag precursor, we investigated the possibility that processing of the gag precursor as well as the gag-pal fusion protein might occur in certain cell lines other than CV-1. We first monitored the RT activity in the culture fluids of various human cell lines infected with the recombinant viruses. SW480, R66, HeLa, HPB-ALL, Molt-4, and MT-4 cells were infected with Vat-gag/PO/or Vat-gag-PO/-fuse at 10 PFU per cell. After incubation at 37’ for 24 hr in the presence of Ara-C, the culture fluid was harvested, clarified by low speed centrifugation, and the RT activity in the supernatant was assayed. Surprisingly, relatively high RT activities were detected in the culture fluids of cells infected with Vat-gag-PO/-fuse, except for fluid from CV-1 and HPB-ALL (Fig. 3). Among these cell lines, SW480 showed the highest RT activity. When culture fluids were mixed, those of infected or uninfected CV1 cells did not reduce the RT activity in culture fluid

Hela R66 HPE-ALL Molt4 MT-4 FIG. 3. Autoradiogram of RT activity in culture fluids. SW480, CV1, HeLa. R66, HPB-ALL, Molt-4, and MT-4 cells were infected with WR or recombinant vaccinia viruses. After incubation at 37’ for 24 hr, culture fluids were examined for RT activity and 10.~1 aliquots of reaction mixtures were spotted onto a nitrocellulose filter.

PARTICLE

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FIG. 4. Time-course of the RT activity. SW480 cells were infected with Vat-gag/pol(left) or Vat-gag-pof-fuse (right), and the RT activity in the culture fluid (open circles) and the cytoplasmic fraction (solid circles) was assayed at the indicated times.

ble form or associated with some structural components, we measured the density of this RT activity. SW480 cells in a 90-mm plate were infected with Vacgag/pal at 20 PFU per cell and incubated at 37” for 24 hr in serum-free medium containing Ara-C. Culture fluid was harvested, clarified by low speed centrifugation, filtered through a nitrocellulose filter of 0.45 pm pore size, concentrated by ammonium sulfate (50% saturation), and layered onto the top of a 1O-40% potassium tat-trate density gradient. After centrifugation at 300,000 g for 2 hr, a visible band appeared at a density of 1.15 g/ml, which contained the highest RT activity (Fig. 5A). This density was very similar to the reported density of HIV particles (Popovic et a/., 1984). Neither culture fluids of WR (Fig. 5B)- nor Vac-gag-polfuse-infected (Fig. 5C) SW480 cells showed such a visible band. In the latter, the RT activity was detected exclusively in the soluble top fractions. Electron microscopy showed that the 1.15 g/ml density fraction with the RT activity contained many round particles with diameters of 100-l 50 nm (Fig. 6) which were also similar in size to HIV particles (for example, Schijpbach eta/., 1984; Stannard eta/., 1987; Chrystie and Almeida, 1988). Enzyme immunoassay using a commercial kit showed that culture fluid of Vat-gag/PO/-infected SW480 cells was antigen-positive up to at least 1OOOfold dilution, indicating that the culture fluid contained HIV antigens equivalent to about 50 to 100 rig/ml of whole HIV virions. Protein analysis of the particle The above Vat-gag/pa/-derived particle fraction was appropriately diluted and centrifuged at 200,000 g for

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2 hr. The pellet was dissolved in a small amount of 100 mM NaCI-10 mM Tris-HCI (pH 7.5)-l mM EDTA (NTE), and part of it was subjected to SDS-PAGE followed by immunoblot analysis (Fig. 7). Serum from an AIDS patient, which contained a full set of antibodies against HIV, detected polypeptides at positions corresponding to the processed major core protein ~24, integrase ~32, and RT ~65 (Fig. 7, left, patient 1 serum). The polypeptide at the position of p24 also reacted with the mouse monoclonal antibody against p24 (anti-p24 in Fig. 71,confirming that the particle fraction contained cleaved gag proteins. On the other hand, only the unprocessed gag precursor ~55 was detected by patient 1 serum within Vat-gag/pa/-infected SW480 cells, as was observed with CV-1 cells (Fig. 7, left). When serum lacking the antibody against p32 integrase was used, the 32K polypeptide in the particle fraction was no longer detected (Fig. 7, left, patient 2 serum), indicating that HIV integrase was properly processed. The presence of ~65 corresponded well to the fact that the RT activity was associated with the particle fraction. Patient 1 serum detected no HIV-specific polypeptide in the fraction prepared from WR-infected SW480 cells in the same manner as used to prepare the Vac-gag/polderived particle fraction (data not shown, see below and Fig. 9). Although Ara-C was included in the culture medium to suppress the replication of the vaccinia virus, we examined by immunoblot analysis the possibility that RT and mature gag proteins were associated with the vaccinia virus (Fig. 7, right). For this analysis, lOOO-fold concentrated culture fluid (which contained 1 mg of protein per milliliter) of Vat-gag/pa/-infected SW480 cells was used as the particle fraction. Anti-WR serum from mice infected with WR virus strongly reacted with 10 pg of purified WR virus, which was included in the analysis as a positive control, but only weakly reacted with the same amount of the particle fraction. Monoclonal antibody against p24 reacted only with the par-ticle fraction. These results strongly suggested that the particles were mainly composed of HIV polypeptides, although traces of vaccinia polypeptides might have been present in the particle fraction. RNA analysis of the particle The above particle fraction in NTE was treated with 2% SDS and deproteinized by phenol extraction. Pellets were obtained by ethanol precipitation and were electrophoresed on a 1% agarose gel for Northern blot analysis in parallel with RNAs prepared from WR-infected and Vat-gag/pal-infected SW480 cells. We found that the particle fraction contained RNA which hybridized with an HIV DNA fragment corresponding to

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Fractbn

numbol

FIG.5. Density gradient centrifugation of the RT activity in culture fluid. Ammonium sulfate-concentrated culture fluid of SW480 cells infected with Vat-gag/pal (A), WR (S), or Vat-gag-pal-fuse (C)was layered onto the top of a 1O-4096 potassium tartrate density gradient. After centrifugation, fractions were collected from the bottom, and portions were concentrated by ammonium sulfate to remove potassium tartrate before assaying the RT activity (open circles). Solid circles and triangles indicate density (g/ml) and the OD at 280 nm, respectively.

the gag and pal genes (Fig. 8). Although the RNA was heterogeneous in size, the largest RNA recovered from the particle fraction was about 5.5 kb long, the same as that of intracellular gag/pa/ mRNA expressed by Vat-gag/pa/. This result suggested that intracellular gag/pal mRNA was packaged in the particles. Protein analysis of SW480 cell culture infected with Vat-gag-pal-fuse As mentioned before, Vat-gag-PO/-fuse-infected SW480 cells did not yield particles with RT activity but produced the enzyme in soluble form (Fig. 5C). In fact, the 65K RT polypeptide was detected in infected culture fluid simply concentrated by ammonium sulfate (Fig. 9, right, ppt-2). The polypeptide was not detected in the sample (Fig. 9, right, ppt-1) prepared from the same culture fluid by the method used to obtain the particle fraction from Vat-gag/PO/-infected SW480 cells, as was the case with WR-infected SW480 cells (Fig. 9, right). The HIV polypeptide pattern within Vac-gag-polfuse-infected SW480 cells was different from that within the same virus-infected CV-1 cells. In addition to the 120K and 32K polypeptides observed in the CV-1 cells (Fig. 2) 51K and 65K polypeptides were clearly demonstrated (Fig. 9, left), suggesting that thegag-pal fusion protein was more efficiently processed in SW480 cells than in CV-1 cells. The reason why polypeptides other than the 65K were not observed in the concentrated culture fluid (Fig. 9, right, ppt-2) is not clear at present.

infection of SW480 cells with Vat-gag/pro or Vat-gag The Vat-gag/pro virus contained the entire gag gene and the 5’ proximal half of the pol gene, which encompassed the complete coding region of the protease but lacked half of the RT region. The Vat-gag virus contained the entire gag gene and 139 codons of the pal gene, which lacked 17 carboxy-terminal amino acids of the protease and the subsequent pal region, Therefore, neither the culture fluids nor the cytoplasmic fractions prepared from SW480 cells infected with these recombinant viruses showed RT activity. This confirmed that the RT activity detected in the preceding experiments was derived from the pal gene. However, potassium tartrate density gradient centrifugation revealed that the culture fluid of SW480 cells infected with these recombinant viruses contained a particle fraction quite similar to that released from the cells infected with Vat-gag/pa/ (data not shown). Although only the ~55 gag precursor was detected in SW480 cells infected with Vat-gag/pro and Vat-gag, as was the case with Vat-gag/PO/-infected SW480 cells (Fig. 9, left), the polypeptide patterns of particle fractions prepared from infected culture fluids differed greatly depending on the recombinant viruses. The Vat-gag-derived particle fraction exclusively contained the uncleaved gag precursor, while the Vat-gag/proderived particle fraction contained both the ~55 precursor and p24 mature gag protein (Fig. 9, right). The processing efficiency of the gag precursor in the Vat-gag/ pro-derived particle fraction seemed to be somewhat

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FIG. 6. Electron micrograph of the particle fraction prepared from culture fluid of Vat-gag&o/-infected fields are presented. Bars indicate 500 nm for A and 100 nm for B, C, and D.

lower than in the Vat-gag/pa/-derived particle fraction, but this result confirmed that the processing of the gag precursor into mature gag proteins was mediated by HIV protease. This result also strongly suggested that the uncleaved gag precursor alone was sufficient for production of the particle.

DISCUSSION In the present study, we demonstrated that HIV-like particles containing active RT, mature gag proteins, and gag/pa/ mRNA were produced from SW480 cells infected with recombinant vaccinia virus carrying the entire gag and pal genes. The size, shape, and density of the particle was very similar to that of HIV, although the particle lacked the env protein. Furthermore, HIV-

SW480 cells. Four electron microscopic

like particle production was also observed in SW480 cells infected with the recombinant vaccinia virus carrying the gag gene alone. These observations resemble a previous finding that a clone of NIH/3T3 cells infected with Moloney murine leukemia virus released C type particles lacking the pal and env proteins (Shields et a/., 1978). Thus, the gag gene product alone appears to be sufficient for production of HIV particles. Mann et al. (1983) demonstrated that when the Moloney murine leukemia virus genome lacked a 350-nucleotide region located between the 5’ proximal splice donor and the initiating methionine codon for the gag precursor it was packaged very poorly into virions, suggesting that this region contained the cis-acting packaging signal. Adam and Miller (1988) showed that this 350-nucleotide region alone was sufficient for the

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FIG. 7. lmmunoblot analysis. (Left) Lysates of WR- or Vac-gag/po/infected SW480 cells and the particle fraction prepared from culture fluid of Vat-gag/pa/-infected SW480 cells were probed with serum from patient 1 containing a full set of antibodies against HIV, serum from patient 2 lacking antibodies against p32 integrase, or the mouse monoclonal ascitic antibody against p24 (anti-p24). (Right) The above particle fraction and WR virus prepared from infected CV1 cells were probed with anti-p24 and pooled sera from mice immunized with WR (anti-WR).

packaging of genomic RNA into the virus particle. Assuming that the region between the 5’ proximal splice donor site and the initiating methionine codon for the gag precursor also serves as the packaging signal for HIV, though this is unproven, it is not surprising that RNA corresponding to the gag and pal genes was packaged in the HIV-like particles, since the HIV DNA we inserted into the vaccinia virus genome contained this putative packaging signal. As to why Vat-gag-pal-fuse-infected SW480 cells failed to produce HIV-like particles despite releasing relatively high RT activity in the soluble form, several explanations seem possible. For example, the failure may have been caused by the loss of the carboxy-terminal half of the pl5 mature gag protein, which is supposed to be an RNA binding protein (Mervis et al., 1988) caused by artificial fusion of the gag and pal genes. When SW480 cells were used, the recombinant vaccinia virus carrying the gag gene followed by the HIV protease gene produced HIV-like particles containing the mature gag protein ~24, whereas the recombinant carrying the gag gene alone yielded HIV-like particles containing only the gag precursor. In both cases, however, the intracellular gag protein was mainly the un-

cleaved precursor. All the findings presented here suggest that the processing of the gag precursor and the assembly of gag proteins into viral particles are very closely related. It seems unlikely that the mature gag proteins are assembled selectively into virus particles. Presumably, the processing of the gag precursor occurs during or after its assembly into the virion. Such a model has been also proposed for the particle formation of type C retrovirus (Bolognesi et al., 1978). In contrast to SW480 cells, CV-1 cells infected with Vat-gag/pa/ did not produce HIV-like particles. Within the cells, the gag precursor was not processed and RT activity was absent, which was consistent with previous findings (Flexner et al., 1988). Moreover, the processing of the artificial fusion protein expressed from Vat-gag-pal-fuse was more effective in SW480 cells than in CV-1 cells. The mechanisms which determine the differences between SW480 and CV-1 cells in producing HIV-like particles and processing the gag-pol fusion protein remain obscure at present. SW480 cells have been reported to produce high titers of HIV by transfection with an HIV infectious molecular clone, although the efficiency of DNA uptake by SW480 cells was the same as that by other human epithelial cell lines (Adachi et a/., 1986). The high efficiency of HIV release from transfected SW480 cells might be explained by the same mechanism which resulted in the high efficiency of particle formation and processing observed here. In a recent communication, Gowda et al.

Or1 +

28s

+

18S-

FIG. 8. Northern blot analysis. A sample prepared from the particle fraction by deproteinization with SDS treatment and phenol extraction followed by ethanol precipitation was electrophoresed on 1% agarose gel in parallel with RNA prepared from Vat-gag/po/- and WRinfected SW480 cells, transferred onto a nitrocellulose filter, and hybridized with a radioactive probe prepared from HIV gag gene. The positions of the ribosomal RNAs, 28 S and 18 S, are indicated. Cri indicates the origin of electrophoresis.

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I SW480 ccl bate

FIG. 9. lmmunoblot analysis. (Left) Lysates of SW480 cells infected with WR or recombinant vaccinia viruses were analyzed using AIDS serum containing a full set of antibodies against HIV. (Right)The particle fractions from Vat-gag/Do/-, Vat-gag/pro-, or Vat-gag-infected SW480 cells, the corresponding fractions prepared from WR-infected or Vat-gag-PO/-fuse-infected (ppt-1) SW480 cell cultures in the same manner as used to prepare the particle fractions, and culture fluid of Vat-gag-PO/-fuse-infected SW480 cells concentrated by ammonium sulfate (ppt-2) were all analyzed using the same serum.

(1989) also reported differences in the processing efficiency of the p55 gag precursor among several human lymphoid cell lines, which they demonstrated using the vaccinia virus expression system. Therefore, it is possible that a certain host cell factor(s) concerning the assembly and/or processing of the HIV gag precursor may exist, and that the activity of this factor may be higher in SW480 cells than in CV-1 cells. The experimental system described here will enable us to obtain much information regarding the processing and assembly of the HIV gag precursor. Furthermore, HIV-like particles produced from Vac-gag/po/-infected SW480 cells may be valuable as immunogens in analyzing both the cellular and humoral immune responses against gag proteins. ACKNOWLEDGMENT We are grateful to Dr. H. Sawada for his guidance in performing the electron microscopic observations and to Dr. K. Hirose for his help in constructing the recombinant vaccinia viruses. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.

REFERENCES ADACHI, A., GENDELMAN, H. E., KOENIG,S., FOLKS,T., WILLEY, R., RABSON, A., and MARTIN, M. A. (1986). Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infections molecular clone. J. Viral. 59,284-291.

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