Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7381-7385, August 1993 Microbiology Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses (capsid release/proteolytic processing/myristoylation) HEINRICH G. G6TTLINGER*, TATYANA DORFMAN*, ERic A. COHENt, AND WILLIAM A. HASELTINE* *Division of Human Retrovirology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; and tDNpartement de Microbiologie et Immunologie, Universite de Montreal, Montreal, Quebec, Canada H3C 3J7 Communicated by Hilary Koprowski, March 15, 1993

ABSTRACT The Vpu protein ofhuman immunodeficiency PR-(vpu+) and HXBc2-PR-(vpu-), the codon for Asp25 of virus type 1 facilitates the release of virus particles from the the HIV-1 protease was changed to a codon specifying surface of infected cells. The ability of the Vpu protein to glutamic acid, as described (7). facilitate release of Gag proteins from retroviruses that lack a Chimeric proviruses were constructed by inserting gag and Vpu-like protein was examined. The results of these experi- pol sequences from HIV-2, visna virus, or Moloney murine ments show that Vpu significantly increases the release of the leukemia virus (Mo-MLV) into HIV-1 (Fig. 1). The HIV-2 Gag proteins of human immunodeficiency virus type 2, visna gag and pol genes were inserted into HXBH10 as a Nar virus, and Moloney murine leukemia virus from HeLa cells. I-Xba I fragment (nt 304-5066), generating HXBH10/ The results indicate that Vpu-mediated enhancement of par- RODgag-pol The HIV-2 Nar I-Xba I fragment was created by ticle release requires neither amino-terminal myristoylation of recombining Nar I-EcoRI and EcoRI-Xba I fragments from the Gag precursor nor cleavage of the Gag precursor by the the ROD27 and ROD35 partial proviral clones (8). The HIV-2 viral protease. The results raise the possibility that Vpu fragment was inserted between the Nar I site in the primer modifies a cellular pathway common to the release of all binding region of HXBH10 (nt 637) and an engineered Xba I retroviruses from the cell surface. site in the HIV-1 vifgene (nt 5228). The visna virus gag-pol region was excised from the Human immunodeficiency virus type 1 (HIV-1) encodes LV1-1KS1 infectious molecular clone (9) as a Nar I-Bgl II several proteins in addition to the gag, pol, and env gene fragment (nt 166-5221) and inserted into HXBH10 between products common to all retroviruses. One of these proteins, nt 637 and 5228 as described above, yielding HXBH10/ Vpu, is unique to HIV-1 (1). Disruption of the vpu reading LVgag.po1. The Mo-MLV gag gene and protease coding frame results in a 5- to 10-fold reduction in the yield of region were excised from the infectious molecular clone NCA progeny virions from infected T-cell lines and an accumula- (10) as an Aat II-Asp718 fragment (nt 367-2862) and inserted tion of virions on the cell surface (2-4). These observations into HXBH10 between an engineered Aat II site (nt 771) 3' to suggest that Vpu acts on a late step in HIV-1 particle the HIV-1 major splice donor site and an Asp718 site at nt morphogenesis. It has also been reported that Vpu facilitates 4153. Klenow DNA polymerase I was used to insert 4 bp at the intracellular transport ofthe Env protein by destabilizing the Asp718 cloning site to prevent translation ofthe HIV-1 int intracellular complexes between Env and CD4, the receptor region, yielding HXBH10/NCAgag. To create variants that for HIV-1 (5). However, the effect of Vpu on HIV-1 particle lack the vpu initiation codon but are otherwise isogenic, a release does not require the expression of the Env glycopro- Nar I-Sal I fragment including the transferred gag and pol tein and is independent ofthe presence or absence ofCD4 (6). sequences was excised from each of the chimeric clones and The ability of Vpu to facilitate virus capsid release in the inserted between the unique Nar I and Sal I sites ofHXBH10- absence of Env may be attributed either to direct interaction vpu-, yielding HXBH10-vpu-/RODgagpoi, HXBH10-vpu-/ of the Vpu protein with HIV-1 internal structural (Gag) LVgagpoi, and HXBH10-vpu-/NCAgag. HXBH10-vpu- is proteins or to the alteration of cellular functions that are identical to HXBH10, except for a point mutation that alters required for effective release of retrovirus capsids. In an the vpu initiation codon (3). To create a second set of vpu- attempt to distinguish between these two possibilities, the chimeric clones, the Nar I-Sal I fragments were inserted into effect of vpu expression on the release of capsids of other the HXBc2 provirus, which does not express a Vpu protein retroviruses that do not normally express a Vpu-like function (3). was examined. The requirement for HIV-1 Gag protein Cell Culture and Transfections. Twenty-four hours prior to processing by the viral protease for Vpu-facilitated release transfection, 106 HeLa (CCL2) or SW480 cells (American was also examined. Type Culture Collection) were seeded into 80-cm2 tissue culture flasks. The cells were transfected with 30 Mg of proviral plasmid DNA by a calcium phosphate precipitation MATERIALS AND METHODS technique (11). In cotransfection experiments, 25 pg of Proviral DNA Constructs. The vpu+ HIV-1 proviral con- proviral DNA and 20 ,g ofvpu expressor plasmid were used. struct HXBH10 has been described (3). HXBH10 was cre- Viral Protein Analysis. Cell cultures were metabolically ated by replacing nt 5372-5934 of the vpu- HIV-1 molecular labeled with [35S]cysteine or [35S]methionine (50 ,Ci/ml; 1 clone HXBc2 with the corresponding segment from the BH10 ,uCi = 37 kBq) from 48 to 60 hr after transfection. Virion molecular clone, thereby introducing a functional vpu gene. release into the labeling medium was monitored by pelleting To generate the protease-defective variants HXBH10- ofparticulate material through a 20% sucrose cushion for 2 hr at 27.000 rpm in a Beckman SW 41 rotor. Pelleted virions The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HIV, human immunodeficiency virus; Mo-MLV, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Moloney murine leukemia virus. 7381 Downloaded by guest on September 26, 2021 7382 Microbiology: Gottlinger et al. Proc. Natl. Acad. Sci. USA 90 (1993) Nar I (Aat II) Asp718 (Xba I) 1 gag r FM+vpu HXBH1 0 A I IPR| RT IN I H UIen SD vpr rev 1 tat 1 --- FMvpu env 'I[ IHULlI FIG. 1. Structure of chimeric provi- HXBH1 O/RODgag-pc . vpr rev 1 ruses. The HXBH10/RODgag-pol and t 7g HIV-2 gag HXBH10/LVgag-j,ol chimeras were gener- I HI////HiV 2 ol ated by replacing HIV-1 gag and pol se- Su tat 1 quences with the corresponding sequences vi FMvpu of HIV-2 or visna virus. The HXBH10/ LTR | . Li [ I env NCAgag chimera harbors Mo-MLV gag HXBH1 0LVgag-pol .vpr rev 1 and protease-coding sequences joined out visngagq of frame to the 3' end of the HIV-1 pol gene. Open boxes represent HIV-1 se- quences; hatched boxes represent HIV-2, SD (Aat Asp718 tat 1 visna virus, or Mo-MLV sequences. Re- (Aat ' ,)U striction enzyme sites used for recombina- L~~~~~T~~~~~K.~L tion are indicated. LTR, long terminal re- HXBH10/NCA gag vpr rev 1 peat; SD, splice donor; PR, protease, RT, _//MLVaQa//PRg reverse transcriptase; IN, integrase. were lysed in Laemmli sample buffer [125 mM Tris, pH Electron microscopic analysis of CD4+ cells infected with 6.8/2% (wt/vol) SDS/4.8% (vol/vol) 2-mercaptoethanol/ HIV-1 has indicated that in the absence of a functional vpu 20% (vol/vol) glycerol], and viral proteins in the pellets were product, virus particles are formed but remain largely at- visualized by electrophoresis through SDS/11.5% polyacryl- tached to the cell surface (4). Similarly, in HeLa cell cultures amide gels. For immunoprecipitation studies, labeled cells transfected with HXBH10-vpu-, large numbers of virions of were lysed in RIPA buffer [140 mM NaCl/8 mM Na2HPO4/2 mostly mature appearance were found close to the cell mM NaH2PO4/1% (vol/vol) Nonidet P-40/0.5% (wt/vol) surface (Fig. 2C). More virus particles appeared to be asso- sodium deoxycholate/0.05% SDS], and virus particles in the ciated with these cells than with HeLa cells transfected with supernatant fractions were disrupted by adding 5 x RIPA the vpu+ strain (Fig. 2B). Moreover, as in T cells infected buffer. HIV-i-encoded proteins were immunoprecipitated by with a vpu- strain of HIV-1 (4), virus particles were fre- using a serum from an HIV-1-infected patient and separated quently found densely packed in intracellular vacuoles in in 11.5% polyacrylamide gels as described (12). The vpu gene HeLa cells transfected with the HXBH10-vpu- construct product was immunoprecipitated with a rabbit serum raised (Fig. 2C). These results indicate that Vpu has a similar, if against a peptide corresponding to aa 73-81 ofthe BH10 Vpu more pronounced, effect on HIV-1 particle production in the protein (1). Electron Microscopy. Transfected HeLa cells were pro- A B cessed for thin-section electron microscopy as described (7). 1 2 3 ...... ,

RESULTS gpl 20- # Effect ofVpu on Release of HIV-1 Capsids from HeLa Celis. The vpu gene product has been shown to enhance the export of virus particles from CD4+ T cells infected with HIV-1 (2, 3). However, the use ofCD4+ T cells is restricted by the need C- for virus replication to yield measurable quantities of virus particles. To permit the analysis of the role of Vpu in mutant viruses independent of their ability to replicate, HeLa cells IN were used as an expression system. HeLa cells were trans- fected with the HXBH10 (vpu+) or HXBH10-vpu- strains, p24 B ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... which differ only at the position of the vpu initiation codon (3). After metabolic labeling with [35S]methionine, virions released into the supematant were pelleted through a 20% sucrose cushion and viral proteins in the pellets were visu- alized by SDS/PAGE. The amount ofviral p24 capsid protein pelleted from cultures transfected with the vpu+ and vpu- p7 strains differed by at least 100-fold asjudged by densitometry (Fig. 2A). The p24 protein was expected to yield the most FIG. 2. Effect of Vpu in HeLa cells. (A) Sedimentation of virus prominent viral protein band due to the number ofmethionine particles from culture supernatants. HeLa cells were transfected residues present in the molecule. Other virally encoded with the vpu+ provirus HXBH10 (lanes 1 and 3) or the HXBH10- proteins were detectable only in pelleted material shed from vpu- provirus (lane 2) and metabolically labeled with [35S]methio- the vpu+ cultures (Fig. 2A, lanes 1 and 3). Despite the striking nine from 48 to 60 hr posttransfection. Virions released into the supernatant were pelleted through 20% sucrose and lysed in Laemmli differences in virus particle yield, the amount and profile of sample buffer, and labeled viral proteins were visualized by SDS/ cell-associated viral proteins were indistinguishable in HeLa PAGE. IN, integrase; p24, capsid protein; p7, nucleocapsid protein. cell cultures transfected with the vpu+ and vpu- strains as (B and C) Transmission electron micrographs of HeLa cells trans- demonstrated by immunoprecipitation from labeled cell ly- fected with the vpu+ provirus HXBH10 (B) or the HXBH10-vpu- sates (Fig. 3A, lanes 1 and 2). provirus (C). (Bar = 250 nm.) Downloaded by guest on September 26, 2021 Microbiology: G6ttlinger et al. Proc. Natl. Acad. Sci. USA 90 (1993) 7383 A B C virus replication. This experimental system was then used to Cells Sup examine the effect of Vpu on the release of capsids of other 1 2 3 4 1 2 3 4 1 2 3 4 retroviruses. Since Vpu appeared to be more efficient when gpl160 - - a -gp120 expressed from the viral genome than when provided in trans gpl20- (5, 6), pairs of chimeric recombinant viral clones were made that differed only in the presence or absence of a vpu initiation codon. The chimeric clones represent HIV-1 with p55 -W -p55 gag and pol sequences replaced by those of HIV-2, visna virus, or Mo-MLV (Fig. 1). The transferred sequences were expected to replace the HIV-1 major splice donor site with that of HIV-2 or visna virus. The Mo-MLV gag gene was p24 - inserted downstream of the HIV-1 major splice donor site to p24 - preserve HIV-1 splicing signals. To test whether the chimeras were able to express proteins synthesized from subgenomic messages, the expression of p17- p17-- the HIV-1 Env glycoprotein was monitored. Significant amounts of gpl60 and gpl20 were immunoprecipitated from the lysate of cells transfected with the parental HXBH10 provirus, the HXBH10/RODgag_pol chimera, or the HXBH10/LVgag.poi chimera (Fig. 4A). By comparison, only FIG. 3. Role ofproteolytic processing in Vpu-mediated enhance- a small amount of HIV-1 Env was detectable in cells trans- ment of capsid release. HeLa cells were labeled with [35S]cysteine fected with the HXBH10/NCAgag construct, possibly reflect- from 48 to 60 hr posttransfection and viral proteins were immuno- ing a paucity of correctly spliced transcripts. The amount of precipitated from the cell lysates (A) or the culture supernatants (B) Env glycoprotein present in the cell lysates was unaffected by with an HIV-1-infected patient serum. In C, virus particles from the presence or absence of a vpu initiation codon. Since culture supematants were pelleted through 20% sucrose and directly efficient HIV-1 env expression requires Tat and Rev activity analyzed by SDS/PAGE after disruption in Laemmli sample buffer. (13), these results indicated that the presence of HIV-2 or The transfected proviruses were HXBH10 (vpu+; lane 1), HXBc2 (vpu-; lane 2), HXBH10-PR- (vpu+; lane 3), and HXBc2-PR- visna gag-pol sequences was compatible with the production (vpu-; lane 4). The band that comigrates with p7 in C is a background of subgenomic transcripts capable of expressing HIV-1 tat, band which is also present in mock samples (see Fig. 5B, lane M). env, and rev genes. The HIV-1 patient serum also recognized the HIV-2 Gag precursor and p26 capsid protein. The amount epithelial cell line HeLa than in lymphoid cells permissive for ofHIV-1 or HIV-2 Gag proteins precipitated was not affected HIV-1 replication. by the presence or absence of a vpu initiation codon. Requirement for Viral Protease Function for Vpu-Mediated The ability ofthe chimeras to express Vpu was analyzed by Capsid Release. To determine whether the effect of Vpu on immunoprecipitation with an anti-Vpu peptide antiserum. A particle release is dependent on proteolytic maturation, the viral protease encoded in the vpu+ HXBH10 provirus and the A B vpu- HXBc2 provirus was inactivated. The amount of the M 1 2 34 5 6 78 1 234 5 p55 Gag precursor was similar in HeLa cells transfected with gpl16- the HXBH10-PR-(vpu+) or HXBc2-PR-(vpu-) proviruses gpl2O - (Fig. 3A, lanes 3 and 4). Processed Gag proteins were not detectable. As observed previously (7), unprocessed Gag precursor, which normally is not released, was exported into the medium in the absence of a functional protease (Fig. 3B, gag Pr E lane 3). However, very little p55 Gag precursor protein was detectable in the supematant fraction of cultures transfected with the PR-/vpu- strain (Fig. 3B, lane 4). The magnitude of the Vpu effect on the export of the unprocessed Gag precur- sor was similar to that on the mature Gag proteins in the presence of a functional HIV-1 protease. In both cases, the effect of Vpu on the amount of gpl20 envelope glycoprotein gag CA E - released into the medium, as determined by immunoprecip- M itation from the supernatant fractions, was much less pro- g. g vpu - nounced than the effect on the Gag proteins (Fig. 3B). This observation is probably best explained by shedding of gpl20 gag MA -_6 into the medium independent of virion release. To determine whether the unprocessed Gag precursor was FIG. 4. Expression of viral proteins in HeLa cells transfected released in a particulate form, [35S]cysteine-labeled particu- with chimeric clones. (A) Immunoprecipitation from [35S]cysteine- late material shed into the supernatant of cells transfected labeled cell lysates with an HIV-1-infected patient serum. The with the PR-/vpu+ and PR-/vpu- strains was centrifuged transfected proviruses were as follows: lane M, mock; 1, HXBH10 through a 20% sucrose cushion, disrupted in Laemmli sample (vpu+); 2, HXBH10-vpu-; 3, HXBH10/RODgagp,,ol (vpu+); 4, buffer, and resolved by SDS/PAGE. The amount of partic- HXBH10-vpu-/RODgag-poI; 5, HXBH10/LVgagp,,ol (vpu+); 6, ulate in the cell was greatly HXBH10-vpu-ILVgag-pol; 7, HXBH10/NCAgag (vpu+); 8, p55 Gag precursor supernatant HXBH10-vpu-/NCAgag. Pr, Gag polyprotein precursor; CA, capsid enhanced by Vpu (Fig. 3C, lanes 3 and 4), confirming that protein; MA, matrix protein. (B) Detection of Vpu protein. Lysates Vpu-facilitated particle release is independent of proteolytic of transfected Hela cells were immunoprecipitated with an anti-Vpu maturation. peptide serum after metabolic labeling with [35S]methionine. The Effect of Vpu on Release of Other Retrovirus Capsids. The transfected proviruses were as follows: lane 1, HXBH10-vpu-; 2, preceding studies demonstrate that the effect of Vpu can be HXBH10 (vpu+); 3, HXBH10/RODgag"o1 (vpu+); 4, HXBH10/ examined by transfection of HeLa cells in the absence of LVgag-pol (vpu+); 5, HXBH10/NCAgag (vPu+). Downloaded by guest on September 26, 2021 7384 Microbiology: Gottlinger et al. Proc. Natl. Acad. Sci. USA 90 (1993) protein ofabout 18 kDa was immunoprecipitated from lysates the vpu- HXBc2/RODgag-po1 construct yielded 15-20 times of cells transfected with HXBH10 (vpu+) but not with less pelletable HIV-2 Gag proteins as measured by densi- HXBH10-vpu- (Fig. 4B, lanes 1 and 2). The amount of Vpu tometry (Fig. SA, lane 4). A similar result was obtained after expressed by the chimeras correlated with the expression of transfection of the HXBH10-vpu-/RODgag-pol construct, Env, consistent with the observation that the Vpu and Env which differs from HXBH10/RODgagpoj only at the position proteins of HIV-1 are synthesized from bicistronic mRNAs of the vpu initiation codon (Fig. SA, lane 5). These results (14). Significant amounts of Vpu were synthesized after demonstrated that the ability of Vpu to enhance particle transfection of the HXBH10/RODgag.poi and HXBH10/ production is not restricted to HIV-1 capsids. LVgagpo, chimeras (Fig. 4B, lanes 3 and 4). By contrast, only The responsiveness ofthe HIV-2 Gag proteins to Vpu may a trace amount of Vpu was detected in cells transfected with have been a consequence ofthe relatively close evolutionary the HXBH10/NCA. chimera (Fig. 4B, lane 5). No Vpu was relationship of HIV-1 and HIV-2. For this reason, the effect detected after transfection of chimeras that lacked a vpu of Vpu on the Gag proteins of visna virus, a more distantly initiation codon (data not shown). related lentivirus (16), was examined. Cotransfection of the To determine the ability of the chimeras to produce virus LV1-1KS1 molecular clone of visna virus and of the vpu particles, [35S]cysteine-labeled particulate material released expression vector SVCMVvpu+ (6) into HeLa cells did not from transfected HeLa cells was pelleted through 20% su- result in release of measurable amounts of virus particles crose. Pelleted virions were lysed in Laemmli sample buffer (data not shown). By contrast, the vpu+ HXBH10/LVga&-po1 and the viral proteins were directly analyzed by SDS/PAGE. construct, which carries the visna gag-pol region in an HIV-1 context, produced significant amounts of visna virus capsids The HXBH10/RODgag_op construct produced Gag proteins upon transfection into HeLa cells (Fig. SB, lanes 1 and 2). As that differed from the HIV-1 Gag proteins in electrophoretic measured by densitometry, the amount of pelletable visna mobility (Fig. 5A, lanes 1 and 3). The mobility was that Gag proteins released from cells transfected with the vpu+ expected for the Gag proteins of HIV-2 (15). Transfection of chimeric virus (Fig. SB, lanes 1 and 2) was 10- to 15-fold higher than that released from cells transfected with the A C 1 2 34 5 12 3 4 56 corresponding vpu- constructs HXBc2/LVgag-pol and 4ItA -p30 HXBH10-vpu-/LVgagpol (Fig. SB, lanes 3 and 4). p26 HIV-1 and visna virus are both members of the lentivirus p24 --O p24- subfamily of retroviruses. To examine whether Vpu could

'I |. enhance the production of particles by the Gag proteins of ..*...... another type of retrovirus, the gag and protease-coding p17-- _ p16 p17 - region of a C-type retrovirus, Mo-MLV, was expressed in an p15 HIV-1 background (Fig. 1). Transfection of the vpu+ con- struct HXBH10/NCAgag into HeLa cells revealed that Mo- B D E MLV capsids were made by the chimera (Fig. SC, lanes 3 and Ml 2 3 4 1 2 3 4 5 1 9 1 A 4). In repeated experiments, about 3-fold more pelletable sr -p30- Mo-MLV Gag proteins were released by the vpu+ construct lanes than the p25-_- . (Fig. SC, 3 and 4) by corresponding vpu- p24-* constructs HXBc2/NCAgag and HXBH10-vpu-INCAgag (Fig. SC, lanes 5 and 6). Since the HXBH10/NCAgag chimera expressed only trace p17-* amounts of Vpu, additional experiments were performed in p16- :. -p15/ which Vpu was provided in trans by the vpu expression vector SVCMVvpu+ (6). As a control, a plasmid identical to p7 -plp0- SVCMVvpu+ except for an altered vpu initiation codon (SVCMVvpu-) was used (6). As previously observed (6), vpu FIG. 5. Effect of Vpu on the production of HIV-2, visna virus, expressed in trans had a significant effect on HIV-1 Gag and Mo-MLV capsids. Hela cells (A-D) or SW480 cells (E) were lanes 1 and Cotransfection ofthe transfected with the indicated proviruses and labeled from 48 to 60 protein export (Fig. SD, 2). hr posttransfection with [35S]cysteine. Particulate material released HXBH10-vpu-/NCAgag chimera and the SVCMVvpu+ ex- into the culture supernatants was sedimented through 20o sucrose pression vector (Fig. 5D, lane 3) resulted in a >10-fold and the viral proteins in the pellets were directly analyzed by increase in pelletable Mo-MLV Gag proteins compared to SDS/PAGE. The transfected proviruses were as indicated below. when the SVCMVvpu- plasmid was used (Fig. SD, lanes 4 (A) Lanes: 1, HXBH10 (vpu+); 2, HXBH10-vpu-; 3, HXBH10/ and 5). RODgag.pfol (vpu+); 4, HXBc2/RODgago,l (vpu-); 5, HXBH10- The effect of Vpu on the export of HIV-1 and Mo-MLV Vpu-jRODag.poi. p26, HIV-2 capsid protein; p16, HIV-2 matrix Gag proteins was also examined in SW480 cells, which like protein. (B) Lanes: M, mock; 1 and 2, HXBH10/LVgag-pol (vpu+); 3, HeLa cells can be transfected with high efficiency (17). The HXBc2/LVgag-pol (vpu-); 4, HXBH10-vpu-/LVga"o1. p25, visna amount of HIV-1 released from capsid protein; p16, visna matrix protein; p14, visna nucleocapsid pelletable Gag proteins protein. (C) Lanes: 1, HXBH10 (vpu+); 2, HXBH10-vpu-; 3 and 4, SW480 cells transfected with the vpu+ strain HXBH10 was 3- HXBH10/NCAgag (vpu+); 5, HXBc2/NCAgag (vpu-); 6, HXBH10- to 5-fold higher than after transfection of the HXBH10-vpu- vpu-INCAgag. p30, Mo-MLV capsid protein; p15, Mo-MLV matrix strain (data not shown). At least a 5-fold increase in Mo-MLV protein. (D) HeLa cells were cotransfected with the vpu expressor Gag protein export was observed when the vpu- chimeras plasmids SVCMVvpu+ (lanes 1 and 3) or SVCMVvpu- (lanes 2, 4, HXBH10-vpu-/NCAgag and HXBc2/NCAgag were cotrans- and 5) and the proviral constructs HXBH10-vpu- (lanes 1 and 2) or fected with the vpu+ expression vector rather than the vpu- HXBH10-vpu-/NCAgag (lanes 3-5). plO, Mo-MLV nucleocapsid control (Fig. 5E). protein. The band which migrates just above the HIV-1 and Mo- MLV nucleocapsid proteins is a background band also present in mock samples as shown in B, lane M. (E) SW480 cells were DISCUSSION cotransfected with the vpu expressor plasmids SVCMVvpu+ (lanes 1 and 3) or SVCMVvpu- (lanes 2 and 4) and the vpu- Mo-MLV gag The results demonstrate that Vpu can significantly enhance constructs HXBH10-vpu-/NCAgag (lanes 1 and 2) or HXBc2/ particle production by Gag proteins from retroviruses as NCAgag (lanes 3 and 4). Lanes 1 and 2 in B, lanes 3 and 4 in C, and divergent as HIV-2, visna virus, and Mo-MLV. These results lanes 4 and 5 in D are duplicate samples. argue against a requirement for a specific interaction between Downloaded by guest on September 26, 2021 Microbiology: G6ttlinger et al. Proc. Natl. Acad. Sci. USA 90 (1993) 7385 Vpu and the Gag proteins of HIV-1, raising the possibility We thank Drs. Henry Slayter and Elisabeth Beaumont for electron that Vpu enhances retroviral budding through modification of microscopy studies and Dr. Katherine A. Staskus for kindly provid- a cellular pathway. ing the LV1-1KS1 molecular clone ofvisna virus. Plasmid pNCA was Earlier work indicated that Vpu facilitated the final release a generous gift from Dr. Stephen P. Goff. H.G.G. was a Sheldon/ of budding virions from the cell surface (4). Since the Andelson American Foundation for AIDS Research Scholar. E.A.C. efficiency of HIV-1 virion release from the cell surface is the recipient of a career award from the National Health Research and Development Program of Canada. This work was supported by appears to be reduced in the absence of proteolytic matura- National Institutes of Health Grant AI29873 to W.A.H. and H.G.G. tion (18, 19), the effect of a block in virion maturation on and by a National Health Research and Development Program/ Vpu-facilitated capsid release was examined. This experi- Medical Research Council Grant to E.A.C. ment showed that Vpu most likely affects the Gag precursor polyprotein rather than processed Gag proteins, since the 1. Cohen, E. A., Terwilliger, E. F., Sodroski, J. G. & Haseltine, effect of Vpu is not diminished in the absence of proteolytic W. A. (1988) Nature (London) 334, 532-534. processing. 2. Strebel, K., Klimkait, T. & Martin, M. A. (1988) Science 241, Another modification of the Gag polyprotein of HIV-1, the 1221-1223. myristoylation of an N-terminal glycine residue, is essential 3. Terwilliger, E. F., Cohen, E. A., Lu, Y., Sodroski, J. G. & for particle production (7, 20). By Haseltine, W. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5163- contrast, the visna Gag 5167. polyprotein lacks an N-terminal glycine residue that could 4. Klimkait, T., Strebel, K., Hoggan, M. D., Martin, M. A. & serve as a myristoylation attachment site (21) and was found Orenstein, J. M. (1990) J. Virol. 64, 621-629. not to be myristoylated (22). Since the production of visna 5. Willey, R. L., Maldarelli, F., Martin, M. A. & Strebel, K. capsids could be enhanced by Vpu, it can be concluded that (1992) J. Virol. 66, 226-234. the effect of Vpu on particle production does not depend on 6. Yao, X. J., Gottlinger, H., Haseltine, W. A. & Cohen, E. A. myristoylation. Also, the visna Gag polyprotein lacks a (1992) J. Virol. 66, 5119-5126. domain comparable to the C-terminal p6 domain ofthe HIV-1 7. Gottlinger, H. G., Sodroski, J. G. & Haseltine, W. A. (1989) Gag precursor (16). Evidently, a p6 domain is not required for Proc. Natl. Acad. Sci. USA 86, 5781-5785. Vpu enhancement of particle production. 8. Clavel, F., Guyader, M., Guetard, D., Salle, M., Montagnier, L. & Alizon, M. (1986) Nature (London) 324, 691-695. The Mo-MLV sequences were the most distant from HIV-1 9. Staskus, K. A., Retzel, E. F., Lewis, E. D., Silsby, J. L., St. in a phylogenetic analysis of 17 retroviruses (23). The limited Cyr, S., Rank, J. M., Wietgrefe, S. W., Haase, A. T., Cook, sequence homology between the gag genes ofthe two viruses R., Fast, D., Geiser, P. T., Harty, J. T., Kong, S. H., Lahti, argues against a requirement for conserved primary sequence C. J., Neufeld, T. P., Porter, T. E., Shoop, E. & Zachow, elements for Vpu-facilitated export. However, there are K. R. (1991) Virology 181, 228-240. examples of functionally related viral capsid proteins that 10. Colicelli, J. & Goff, S. P. (1988) J. Mol. Biol. 199, 47-59. share a common tertiary structure in the absence of signifi- 11. Cullen, B. R. (1987) Methods Enzymol. 152, 684-704. cant primary sequence homology (24). Therefore, conforma- 12. Dayton, A. I., Sodroski, J. G., Rosen, C. A., Goh, W. C. & tional determinants rather Haseltine, W. A. (1986) Cell 44, 941-947. than primary sequence elements 13. Feinberg, M. B., Jarrett, R. F., Aldovini, A., Gallo, R. C. & may be involved in Vpu-mediated enhancement of particle Wong-Staal, F. (1986) Cell 46, 807-817. production. 14. Schwartz, S., Felber, B. K., Fenyo, E. M. & Pavlakis, G. N. Vpu is unique to HIV-1. Why then are the Gag proteins of (1990) J. Virol. 64, 5448-5456. other retroviruses also responsive to Vpu? It is conceivable 15. Brun-Vezinet, F., Rey, M. A., Katlama, C., Girard, P. M., that the vpu gene product affects a common intracellular Roulot, D., Yeni, P., Lenoble, L., Clavel, F., Alizon, M., transport pathway that is followed by different retroviral Gag Gadelle, S., Madjar, J. J. & Harzic, M. (1987) Lancet i, proteins. The release ofMo-MLV capsids from cells could be 128-132. inhibited by treatment with monensin, which blocks intra- 16. Stephens, R. M., Casey, J. W. & Rice, N. R. (1986) Science 231, 589-594. cellular vesicular transport (25). Immunofluorescence stud- 17. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, ies support the hypothesis that the Mo-MLV Gag proteins are R., Rabson, A. & Martin, M. A. (1986) J. Virol. 59, 284-291. transported to the cell membrane via association with the 18. Peng, C., Ho, B. K., Chang, T. W. & Chang, N. T. (1989) J. outer leaflet of intracellular vesicles (25, 26). However, the Virol. 63, 2550-2556. insensitivity of HIV-1 particle production to treatment with 19. Schatzl, H., Gelderblom, H. R., Nitschko, H. & von der Helm, brefeldin A suggests that the HIV-1 Gag proteins are not K. (1991) Arch. Virol. 120, 71-81. routed to the plasma membrane by vesicular transport via the 20. Bryant, M. & Ratner, L. (1990) Proc. Natl. Acad. Sci. USA 87, Golgi complex (27). 523-527. Another possibility is that Vpu can substitute for host cell 21. Towler, D. A., Eubanks, S. R., Towery, D. S., Adams, S. P. & Glaser, L. (1987) J. Biol. Chem. 262, 1030-1036. factors that provide a Vpu-like activity. This hypothesis is 22. Rein, A., McClure, M. R., Rice, N. R., Luftig, R. B. & supported by the observation that the response to Vpu differs Schultz, A. M. (1986) Proc. Natl. Acad. Sci. USA 83, 7246- dramatically in different cell lines. The vpu gene product is 7250. virtually required for HIV-1 particle production in the HeLa 23. McClure, M. A., Johnson, M. S., Feng, D. F. & Doolittle, CCL2 line (6). By contrast, in COS-7 monkey cells, HIV-1 R. F. (1988) Proc. Natl. Acad. Sci. USA 85, 2469-2473. particle production is high even in the absence of Vpu when 24. Rossmann, M. G. & Johnson, J. E. (1989) Annu. Rev. Bio- normalized for the total amount of Gag protein synthesized, chem. 58, 533-573. and HIV-1 particle production is not significantly enhanced 25. Hansen, M., Jelinek, L., Whiting, S. & Barklis, E. (1990) J. by Vpu (28). This striking difference may be explained by the Virol. 64, 5306-5316. 26. Jones, T. A., Blaug, G., Hansen, M. & Barklis, E. (1990) J. presence of a cellular Vpu-like activity in COS-7 but not in Virol. 64, 2265-2279. HeLa cells. Cell-type dependency ofthe requirement for Vpu 27. Pal, R., Mumbauer, S., Hoke, G. M., Takatsuki, A. & Sam- could be due to differential expression of factors that sup- gadharan, M. G. (1991) AIDS Res. Hum. Retroviruses 7, 707- press rather than stimulate retroviral particle formation. In 712. this case, Vpu might serve to overcome such repressive 28. Gottlinger, H. G., Dorfman, T., Sodroski, J. G. & Haseltine, factors. W. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3195-3199. Downloaded by guest on September 26, 2021