Virus Research, 24 (1992) 197-210 0 1992 Elsevier Science Publishers
VIRUS
197 B.V. All rights reserved
0168.1702/92/$05.00
00796
Morphogenesis
Gerald
of recombinant core particles
HIV-2 gag
Voss ‘, Frank Kirchhoff ‘, Sigrid Nick ‘, Dieter Moosmayer Hans Gelderblom ’ and Gerhard Hunsmann a
’ Deutsches
Primatenzentrum,
(Received
Abteilung Virolo@e und Immunologic, ’ Robert-Koch-lnstitut, Berlin, Germany
6 February
1992, revision
received
and accepted
GGttingen, Germany
18 March
‘, and
1992)
Summary
The gag-pol coding region of the HIV-2 nEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VW. These recombinant VV encoded either the whole gag-pal region or the gag gene including the proteasecoding region of the pol gene or the gag gene truncated at its 3’-end or only the pal gene. The HIV-2,,, gag precursor ~55, its mature cleavage products p24 and p17 as well as the pal reverse transcriptase CRT) p66 were detected in VV-infected CV-1 cells. The’p55 and two intermediate cleavage products p40 and ~35 were myristilated. Comparison to lysates of permanently HIV-2,,,-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3’-end of the gag gene coding for the p16 protein is essential for the
Correspondence to: Gerald Voss, Deutsches Kellnerweg 4, D-3400 GGttingen, Germany.
Primatenzentrum,
Abteilung
Virologie
und Immunologic,
198
formation of complete HIV-2 assembly of the viral core. HIV-2;
Gene
gag; Gene
particles
pol; Recombinant
and
that
the
VV; Particle
pal
proteins
support
the
assembly
Introduction The gag gene of human (HIV) and simian (SIV) immunodeficiency viruses codes for the major viral structural proteins. These proteins are processed from a precursor molecule (~55) into the core protein p24 and the inner coat protein ~17. The processing is mediated by the viral protease encoded at the 5’-terminus of the pol gene (Kramer et al., 1986; Debouck et al., 1987) in the gag-pal overlapping region. The expression of the pal gene occurs by frame shifting from the gag to the pal open reading frame (Jacks et al., 1988). The role of the viral protease for proper processing of the gag precursor has been investigated using recombinant proteins derived from various expression systems. Processing of the gag precursor by the viral protease was first reported in yeast cells by expression of the whole gag-pal region (Kramer et al., 1986). Coexpression of the viral protease and the gag precursor showed that the protease is responsible for the processing (Debouck et al., 1987; Overton et al., 1989). The processing of the gug precursor of HIV-l and HIV-2 expressed in mammalian cell lines depended on the host cell line (Flexner et al., 1988; Gowda et al.. 1989; Mars et al., 1990; Moosmayer et al., 1991). Recombinant viral particle structures were observed in mammalian cells infected with recombinant VV coding the HIV-l gag gene (Hu et al., 1990; Shioda and Shibuta, 1990) and with a gag-coding simian virus 40 replacement vector (Smith et al., 1990). These particles had an electron-dense ring-like shape of around 100 nm in diameter. Similar particles were produced in insect cells when infected with recombinant baculoviruses expressing the gug genes of HIV-l (Gheysen et al., 1989; Overton et al., 19891, HIV-2 (Luo et al., 19901 and SIVMA,. (Delchambre et al., 1989). Moreover, particles containing both HIV-l gag and enl’ proteins were observed after coinfection of mammalian cells with two recombinant VV containing the gag and the em’ gene (Haffar et al., 1990; Vzorov et al., 1991). In all cases the uncleaved gag precursor was able to form immature circular particles. Assembly to regular HIV-like core structures was only detected when processed p24 was present (Smith et al., 1990; Haffar et al., 1990; Karacostas et al., 1989). However, little was known about the regions of the gag and pal gene responsible for a proper particle assembly. In this report, we have examined the properties of recombinant gag and pal et al., 1990) expressed with recombinant VV. We proteins of HIV-2 nEN (Schneider showed the efficient expression of HIV-2 UEN gag and pal proteins with recombinant VV in CV-1 cells. In the presence of the protease-coding region of the pal gene, the gug precursor p55 was cleaved into the mature p24 and ~17 proteins.
199
Furthermore, an uncleaved gag p.59 precursor was processed after coexpression of the pal gene with respective VV. Using electron microscopy different states of virus maturation were detected. Early budding structures (VVgag >, circular immature particles (VVgag’) and mature HIV cores (VVgagpol) were formed by the recombinant gag proteins. The reported findings indicate that HIV-2 gag proteins are processed by the viral protease and that they are capable of self-assembly into immature retroviral particles and occasionally to mature core structures similar to HIV-l gag proteins. Furthermore, the gag p16 protein seems to be essential for complete budding.
Materials
and Methods
Cells and LTiruses in Molt 4 clone 8 The HIV-2 nEN isolate (Schneider et al., 19901 was propagated cells (Kikukawa et al., 1986) and used as a control in Western blot analyses of the recombinant gag and pol proteins. The HIV-2,,, genes for the construction of recombinant VV were derived from the proviral MK6 clone of HIV-2,,, (Kirchhoff et al., 1990a). Purified VV-DNA for in vivo recombination was made from VV wildtype (wt) strain Copenhagen. The recombination was carried out in CV-1 cells (Jensen et al., 1964) infected with the temperature-sensitive mutant VVts7 (Condit et al., 19831, which grows at 33°C and is inhibited at 39S”C. The selection for thymidine kinase (TK-)-defective VV with bromodeoxyuridine (BrDU) was performed in TK- mouse L-cells (LM (TK-I) (Kit et al., 1963). Recombinant rlaccinia [iruses The HIV-2,,, gag and pol genes were cloned into the VV insertion plasmid pTG186poly (Kieny et al., 1986). The plasmid contained the VV TK gene interrupted by the VV 7.5K promoter and a polylinker. Four different fragments of the HIV-2 nn,, genome (Kirchhoff et al., 1990b) (Fig. la) were inserted into the plasmid: (i) the gag gene truncated at its 3’-end overlapping the pol reading frame (gagg> (nucleotides 1107-25921, (ii) the gag gene including the protease-coding region of the pol gene (gag’) (1107-32121, (iii) the complete gag-pol region (gagpol) (1107-5758) and (iv> the pol gene (poll (2323-5758). The gag+ and gag-pol genes were subcloned into pTG186poly without further modification. Gag- was generated by excising most of the protease-coding region (downstream from 2592) from gag+ in pTG186gag+ and religation of the plasmid. This resulted in the extension of the reading frame into plasmid derived sequences coding for additional 85 amino acids. For subcloning in pTG186poly and expression of the pol gene, a Sal1 restriction site and an ATG start codon were incorporated at positions 2317 and 2323 of the HIV-2,,, sequence. Mutagenesis was essentially performed as described by Fritz et al. (1988), using the oligonucleotide 5’-
pal
\
:-’
;,
I
’
Fig. 1. Structure of HIV-2,,, gcr,e and &coding recombinant VV. (a) Four different regions of the ,qzg and pal open reading frames of the proviral HIV-2u,, clone were inserted into the VV insertion vector pTG186poly. The genetic structure of the resulting recombinant VV is shown. The crossed region of VVgug indicates the translated part of pTG186poly downstream the truncated gag gene and comprises 85 amino acids. The Sal1 * restriction site was generated by oligonucleotide-directed mutagenesis to generate VVpol (see Materials and Methods). (b) The resulting budding structures and particles after expression of the different recombinant VV are shown schematically.
GCAGCCCTGTCTTCATGGTCGACGGCACTGTCTTGC-3 ’ (mismatches are underlined) spanning bases 2303 to 2339 of the HIV-2nEN sequence. For in vivo recombination of the insertion plasmids 1 X 10” CV-1 cells were infected with 1 pfu per cell of VVts7. The VV were allowed to grow for 2 h at 33°C. The cells were washed once with serum-free Dulbecco’s modified Eagle medium (DMEM) and transfected with 250 ~1 of calcium-phosphate precipitate containing 200 ng insertion plasmid and 1 pug VVwt DNA. After 1 h, the precipitate was removed and the cells were washed twice with serum-free DMEM. Subsequently, the cells were incubated at 395°C in DMEM with 10% fetal calf serum (FCS) and after 48 h, the cultures were frozen and thawed twice. The supernatants were used to infect LM (TK-) cells and recombinant viruses were selected in DMEM containing 10% FCS, 0.5% agar noble (Difco, Detroit, USA), 0.5% LMP-agarose (BRL, Gaithersburg, USA) and 150 pg per ml BrDU. Well isolated plaques were recovered and recombinant VV were expanded on CV-1 cells. The plaque purification was repeated twice. The molecular structure of the recombinant VV was determined by restriction endonuclease digestion and Southern blot analysis. Detection of recombinant proteins To analyze the recombinant proteins by Western infected with 1 pfu per cell of recombinant VV. After
blotting, CV-1 cells were 24 h the cells were frozen
201
once and pelleted. The pellets were suspended in low salt extraction buffer (LSEB) (10 mM Tris, 0.14 M NaCl, 2 mM MgCl,, 1 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride and 0.5% (v/v) Nonidet P-40) and incubated for 15 min on ice. The cell debris were pelleted at 1000 X g and 25 pg protein from the supernatants were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in a 9-16% gradient gel. Proteins were transferred to nitrocellulose sheets at 350 mA overnight. The sheets were saturated with 3% dry milk powder in phosphate buffered saline (PBS), (120 mM NaCl, 17 mM Na,HPO,, 3 mM KH,PO,, pH 7.2) and an macaque antiserum (Stahl-Hennig et al., 1990) or monospecific HIV-2 a,,-specific rabbit antisera to HIV-l ~17, SIV,,, p24 or HIV-l RT were added in PBS with 0.2% NP-40 for 2 h at 37°C. After three washes, an alkaline phosphatase-conjugated goat anti-human or anti-rabbit antiserum (Dianova, Hamburg, Germany) was incubated for 1 h at 37°C. After four washes with TBS (20 mM Tris, 500 mM NaCl, pH 7.5) and a final wash with carbonate buffer (100 mM NaHCO,, 1 mM MgCl,, pH 9.8), the blots were developed with p-nitroblue tetrazoliumchloride (Biorad, Miinchen, Germany) and 5-bromo-4-chloro-3-indolyl-phosphatetoluidine (Sigma, Deisenhofen, Germany). (“H]myristic acid labeling 1 x 10h CV-1 cells were infected with 1 pfu of recombinant VV per cell and incubated for 16 h at 37°C in DMEM supplemented with 10% delipidated FCS. Then 50 PCi [“Hlmyristic acid (Amersham, Braunschweig, Germany) were added for 4 h and the cells were lysed in LSEB. The samples were centrifuged at 1000 x g, the supernatants were removed and the pellets were suspended in PBS. Supernatants and pellets were subjected to SDS-PAGE. The gel was fixed, amplified and dried. Labeled proteins were visualized by autoradiography. Electron microscopy Recombinant VV were used to infect CV-1 cells at 1 pfu per cell. After 24 h, when complete cytopathic effect was visible the cells were fixed in situ with 2.5% (v/v) glutardialdehyde in PBS. Cells were scraped off the plastic surface, sedimented at 275 x g and post fixed with 1% (w/v) 0~0,. After agar block enclosure cells were treated with 1% (v/v) tannic acid and processed further for Epon embedding using routine procedures (Gelderblom et al., 1987). Ultrathin sections were post-stained using Pb-citrate and examined at a Zeiss EM 10A electron microscope.
Results Recombinant HIV-2,,, in CV-1 cells
gag and pol proteins were expressed and efficiently processed
The recombinant HIV-2,,, gag and pol proteins infected with respective VV. Lysates of VV-infected
were expressed in CV-1 cells CV-1 cells were compared to
MI
IIIIIII
23456
i:.
366 -p59 -p55
-p24 ” pl7
Fig. 2. Western blot analysis of recombinant HIV-2 ,3,YAgcr,g XXI pal proteins expressed with recombinant VV. Lysates of CT-1 cells infected with VVgag(lane 2), VVgag’ (lane 3). VVgagpol (lane 4). VVpol (lane 5) or VVwt (lane 1) and HIV-I,,,:,-infected Molt-4 clone X cells (lane 6) were subjected to Western blot analysis using on HlV-2 ,,,,N-spccific macaque antiserum. Prestained protein markers (lane Mf had the following molecular wrights: 180, 116.84. 5X.4X.5and 36.5 kDa.
HIV-2,,,-infected Molt 4 clone 8 cells by Western blotting using an HIV-2,,,specific macaque serum (Fig. 2). Both, the recombinant VVgagpol and VVgag.+ included the protease-coding region of the pal gene. In CV-1 cells infected with these two recombinants. the gng precursor $5 was efficiently processed into the mature p24 and ~17. Both proteins exhibited the same elcctr~)phorctic mobility as the recombithe corresponding proteins in HIV-2 HE;N-infected cells. Furthermore, nant p24 and p17 as well as the pSS precursor were stained by respective monospecific rabbit antisera (data not shown). Radiolabeling of VV-infected CV- 1 cells with [‘Hjmyristic acid revealed that the recombinant pS5 gag precursor was myrist~lated (Fig. 3). Additionally, the expression of the recombinant g~~:ugproteins yielded some intermediate proteins, which did not occur in HIV-2,,,,-infected
203
Fig, 3. Myristilation of recombinant gag proteins. CV-1 cells were infected with VVwt (lane l), VVgag+ (lane 2), VVgag(lane 3) or VVgagpol (lane 4) and labeled with [“Hlmyristic acid (see Materials and Methods). After cell lysis the pellets were resuspended in PBS and separated electrophoretically. An autoradiography of the gel is shown.
cells. Two prominent bands at 40 and 35 kDa and an additional protein at 26 kDa were detected. Analysis with monospecific antisera revealed that the 26 kDa protein contained the p24 protein, while the other proteins comprised both, p24 and ~17 portions (data not shown). Like the ~5.5 precursor, the intermediate proteins at 40 and 35 kDa were myristilated (Fig. 3). The recombinant VVgagpol contained the complete gag and pol reading Western blot analysis showed that three additional proteins frames of HIV-2,,,.
204
were detectable in the respective cell lysates compared to VVgag+ (Fig. 2). One protein of 66 kDa was also present in HIV-2,,,infected cells and was stained by an RT-specific antiserum (data not shown). Two other proteins of 80 and 95 kDa were not found in HIV-2,,,-infected cells. The three proteins at 66, 80 and 95 kDa were also detected after infection of CV-1 cells with VVpol. Furthermore, six other recombinant proteins at 30, 53, 85, 90, 110 and 120 kDa were expressed in VVpol-infected cells. The protease-coding precwsor molecule
region of the pol gene is essential for processing
of the gag
To prove that the processing of the gag precursor p55 was mediated by the viral protease, a recombinant VV with a deletion at the 3’-end of the gag gene
I23 456-78910 I I I I I I I I I
Ml
I
I
P59 - p55 _
- p24
Fig. 4. Proteolytic cleavage of the pSY grog precursor by the viral protease. CV-I cells were infected with VVwt (lanes l-3) or VVpol (lanes 4-h) and after 4 h coinfected with 1 (lanes 1. 4), 5 (lanes 2, 5) or 20 (lanes 3, 6) pfu of VVgag-. After 24 h, cells were harvested and cytoplasmic lysates were subjected to SDS-polyacrylamide gel electrophoresis and subsequent Western blotting with a p24-specific rabbit antiserum. The lysates were compared to those of VVgag+ (lane 7). VVpol (lane 8) and VVgag(lane 9).infected CV-I cells and to HIV-2,,,:, infected Molt 4 clone X cells (lane 10).
20s
removing a part of the protease-coding region (VVgagg) was constructed. When this VVgag- was used to infect CV-1 cells, a gag precursor of 59 kDa was expressed (Fig. 21, which was myristilated like the recombinant p55 (Fig. 3). The high molecular weight of this precursor was due to expression of flanking regions of pTG186poly coding for additional 85 amino acids (Fig. la>. Although the precursor contained p24 and ~17, no processing into the mature proteins was observed. This shows that removal of the protease-coding region of the pol gene abolished the processing of the gag precursor.
Fig. 5. Retroviral were generated
particles formed by recombinant gag proteins. after infection with VVgagpol (e,f), VVgag+ represents 100 nm.
Electron micrographs of CV-1 cells (c,d) and VVgag(a,b). The bar
The recombinant protease of Wpol cleaL,es the gag precursor For further evaluation of the role of the viral protease, CV-1 cells were coinfected with VVgagand VVpol or VVwt. In cells coinfected with VVgag and VVwt the gag precursor was not processed, whereas coinfection with VVgag and VVpol resulted in production of small amounts of p24 (Fig. 4) depending on the quantity of ~59 precursor. This is a second evidence for the cleavage of the gag precursor by the viral protease. Formation of recombinant retroriral particles Finally, the recombinant gag proteins were investigated for their ability to self-assembly into retroviral particles and whether this assembly was depending on a proper processing of the gag precursor. Electron microscopy revealed that in CV-1 cells infected with VVgagtypical budding structures occurred. Virus formation did not proceed further and was apparently stopped at an early state in assembly (Fig. 5a,b). Expression of VVgag+ resulted in formation of particles with approximately 100 nm diameter, which were released in a normal way representing immature lentiviruses, except for the lack of envelope surface protein knobs. However, the released particles did not mature further (Fig. 5c,d). Fig. 5e,f show representative particles observed in cells infected with VVgagpol. Most of the cell-associated structures represent immature ~55 particles. Only a few mature virions were detected containing condensed mature tubular cores (Fig. 5f). A schematic drawing of the budding structures is shown in Fig. lb.
Discussion were expressed in CV-1 cells infected with The gag and pol genes of HIV-2,,, recombinant VV. The cleavage pattern of the gag precursor and the function of the viral protease was found to be similar to HIV-l. The recombinant proteins were identified with specific antisera and the gag precursor was shown to be myristilated. Deletion of the protease-coding region abolished processing of the gag precursor. The processing could be reconstituted by expression of the pol gene in VVgag--infected cells. Formation of budding structures occurred with unprocessed gag precursor and immature circular particles were observed after infection of CV-1 cells with VVgag+. Expression of VVgagpol yielded a few mature HIV cores. The expression of the gag and pol proteins with different recombinant VV yielded precursor and processed HIV-2 nEN proteins of the same size as they were detectable in HIV-2 a,,-infected Molt 4 clone 8 cells. In addition, monospecific antisera showed a reaction pattern with the recombinant proteins similar to that observed with HIV-2 nEN- infected cells. From these findings we conclude that the recombinant proteins produced by recombinant VV are identical to the naturally
207
occurring HIV-2 proteins. Beside these expected proteins, various additional gag intermediates and several &-related proteins were synthesized in the CV-1 cells infected with recombinant VV. These proteins were artificial products of the expression system. Similar differences to HIV-infected cells were found when HIV-l gag proteins were expressed with recombinant VV (Hu et al., 1990). The functional activity of the HIV protease also seems to depend on the cell type used for expression (Mars et al., 1990; Moosmayer et al., 1991) and on other factors, which remain unclear. For example, expression of the whole gag-pol region of HIV-l resulted in the production of mature gag proteins (Gheysen et al., 1989; Karacostas et al., 1989, Gowda et al., 1989), while other investigators did not observe a specific processing (Flexner et al., 1988; Shioda and Shibuta, 1990). The two HIV-2,,, gagpol and gag+ constructs showed efficient processing in several cell lines like Molt 4 clone 8, CEM, macaque B lymphoblastoid cells and BHK-21 cells. One exception was observed in LM (TK-) cells, where no processing was detected (data not shown). When the protease-coding region was deleted from the gag+ construct, the resulting gag precursor was not processed. Similar observations were reported for HIV-l gag precursors (Hu et al., 1990). Thus, absence of the viral protease is responsible for the lack of proteolytic cleavage. The coinfection of CV-1 cells with VVgagand VVpol showed a partial processing of the gag precursor ~59. This indicates that a functional protease was expressed by VVpol. Furthermore, this result reveals that the viral protease does not only cleave the adjacent gag precursor comprised in the large gagpol molecule as suggested by the results of Kramer et al. (1986), Gowda et al. (19891, Gheysen et al. (1989), Karacostas et al. (1989) and Hu et al. (1990), for HIV-l, but that it acts also on other gag precursors. This is consistent with observations of coexpression of the HIV-l protease and the gag precursor in E. coli (Debouck et al., 1987) and with baculoviruses in insect cells (Overton et al., 1989). Our electron microscopy observations suggest that the morphogenesis of HIV-2 core particles proceeds in several distinct steps. Other investigators have shown that the first step in immunodeficiency virus core assembly is the membrane anchorage of the gag precursor mediated by the myristic acid at its N-terminal end (Gheysen et al., 1989; Bryant and Ratner, 1990). Subsequently, budding structures are formed and particles with a circular electron-dense ring are released from the cell surface (Overton et al., 1989; Hu et al., 1990; Shioda and Shibuta, 1990; Gheysen et al., 1989; Luo et al., 1990; Delchambre et al., 1989). Our findings suggest that the C-terminal part of the gag precursor is also involved in the formation of complete particles. The gag precursor truncated in the pl6-coding region assembled only into incomplete budding structures. This phenomenon does not seem to be due to the lack of protease activity because the uncleaved gag precursor was repeatedly shown to form complete particles (Hu et al., 1990; Shioda and Shibuta, 1990; Gheysen et al., 1989; Luo et al., 1990; Delchambre et al., 1989). Deletion of the C-terminal portion of the SIV,,o gag precursor completely abolished particle formation and led to protrusions of the cell surface (Gheysen et al., 1989). Furthermore, mutations in the C-terminal p6 gag protein of HIV-l
20x
prevented the release of budded particles (Gottlinger et al., 1991). Hoshikawa et al. (1991) observed a complete lack of budding when the HIV-l p9 gag protein was deleted. These findings support our assumption of the requirement of p16 for complete particle formation, although it can not be excluded that the adjacent plasmid-derived portion of the HIV-2 nEN ~59 precursor may have prevented complete budding. The final step in particle assembly is the formation of the viral core consisting of ~24. A prerequisite for this process is the cleavage of p24 from the precursor by the protease (Gelderblom, 1991). In fact, mature HIV particles produced by recombinant expression systems were only observed when processed p24 was present (Overton et al., 1989; Haffar et al., 1990; Karacostas et al., 1989). The recombinant HIV-2 nEN proteins expressed by VVgag+including p24 did not assemble to mature particles. This was only observed when the whole gag-pol region was expressed. Immunoprecipitation of radiolabeled recombinant gag proteins revealed that higher amounts of cleaved p24 were produced by VVgagpol compared to VVgag+ (data not shown). Furthermore, the processing of the gug precursor from VVgagpol was not as efficient as in chronically HIV-2,,,-infected cells. Therefore the expression of the pal gene actively supports the formation of mature particles, possibly facilitating a more efficient processing of the gag precursor. Since HIV recombinant gag particles are immunogenic in rabbits (Luo et al., 1990; Vzorov et al., 1991) and additional viral proteins like the RT (Shioda and Shibuta, 1990) or the envelope glycoprotein (Haffar et al., 1990; Vzorov et al., 1991) can be included, these particles offer new perspectives for the development of vaccines against AIDS. In this context, it is of interest that the HIV-2,nN gag particles described in this study are derived from a human immunodeficiency virus able to infect macaques (Stahl-Hennig et al., 1990). Thus, the macaque model offers the opportunity to investigate the potential of HIV-2,,, gag particles as an AIDS vaccine.
Acknowledgements The authors would France) for supplying doctoral thesis of G.V.
like to thank Dr. M.-P. Kieny (Transgene, us with pTG186poly. This work contains
Strasbourg, parts of the
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209 Delchambre, M., Gheysen, D., Thines, D., Thiriart, C., Jacobs, E., Verdin, E., Horth, M., Burny, A. and Bex, F. (1989) The gag precursor of simian immunodeficiency virus assembles into virus-like particles. EMBO J. 8, 2653-2660. Flexner, C., Broyles, S.S., Earl, P., Chakrabarti, S. and Moss, B. (1988) Characterization of human immunodeficiency virus gag/pal gene products expressed by recombinant vaccinia viruses. Virology 166, 339-349. Gelderblom, H.R., Hausmann, E.H., Gzel, M., Pauli, G. and Koch, M.A. (1987) Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156, 171-176. Gheysen, D., Jacobs, E., De Foresta, F., Thiriart, C., Francotte, M., Thines, D. and De Wilde, M. (1989) Assembly and release of HIV-l precursor pr55%“” virus-like particles from recombinant baculovirus-infected insect cells. Cell 59, 103-l 12. Gottlinger, H.G., Dorfman, T., Sodroski, J.G. and Haseltine, W.A. (1991) Effects of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc. Natl. Acad. Sci. USA 88, 3195-3199. Gowda, S.D., Stein, B.S., Steimer, K.S. and Engleman, E.G. (1989) Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with recombinant vaccinia virus. J. Viral. 63, 1451-1454. Haffar, O., Garrigues, J., Travis, B., Moran, P., Zarling, J. and Hu, S.-L. (1990) Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system. J. Viral. 64, 2653-2659. Hoshikawa, N., Kojima, A., Yasuda, A., Takayashiki, E., Masuko, S., Chiba, J., Sata, T. and Kurata, T. (1991) Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study. J. Gen. Vir. 72, 2509-2517. Hu, S.-L.. Travis, B.M., Garrigues, J., Zarling. J.M., Sridhar, P., Dykers, T., Eichberg, J.W. and Alpers, C. (1990) Processing, assembly and immunogenicity of human immunodeficiency virus core antigens expressed by recombinant vaccinia virus. Virology 179, 321-329. Jacks, T., Power, M.D., Masiarz, F.R., Luciw, P.A., Barr, P.J. and Varmus, H.E. (1988) Characterization of ribosomal frameshifting in HIV-l gag-pol expression. Nature 331. 280-283. Jensen, F.C., Giradi, A.J., Gilden, R.V. and Kaprowski. H. (1964) Infection of human and simian cultures with Rous Sarcoma virus. Proc. Natl. Acad. Sci. USA 52, 53-62. Karacostas, V., Nagashima, K., Gonda, M.A. and Moss, B. (1989) Human immunodeficieny virus-like particles produced by a vaccinia virus expression vector. Proc. Natl. Acad. Sci. USA 86, 8964-8967. Kieny, M.-P., Rautmann, G., Schmitt, D., Dott, K., Wain-Hobson, S., Alizon, M., Girard, M., Chamaret, S., Laurent, A., Montagnier, L. and Lecocq, J.P. (1986) AIDS virus env protein expressed from a recombinant vaccinia virus. Bio/Technology 4, 790-795. Kikukawa, R., Koyanagi, Y., Harada, S., Kobayashi. N., Hatanaka, M. and Yamamoto, N. (1986) Differential susceptibility to the acquired immunodeficiency syndrome retrovirus in cloned cells of human leukemic T-cell line Molt-4. J. Viral. 57, 1159-1162. Kirchhoff, F., Jentsch, K.D., Stuke, A., Mous, J. and Hunsmann, G. (1990a) Genomic divergence of an HIV-2 from a german AIDS patient probably infected in Mali. AIDS 4, 847-857. Kirchhoff, F., Jentsch, K.D., Bachmann, B., Stuke, A., Laloux, C.. Luke, W., Stahl-Hennig, C.. Schneider, J., Nieselt, K., Eigen, M. and Hunsmann, G. (1990b) A novel proviral clone of HIV-2: biological and phylogenetic relationship to other primate immunodeficiency viruses, Virology 177, 305-311. Kit. S., Dubbs, D.R., Piekarski, L.J. and Hsu, T.C. (1963) Deletion of the thymidine kinase activity from L cells resistant to bromodeoxyuridine. Exp. Cell Res. 31, 297-312. Kramer, R.A., Schaber, M.D., Skalka, A.M., Ganguly, K., Wong-Staal, F. and Reddy, E.P. (1986) HTLV-III gag protein is processed in yeast cells by the virus pollprotease. Science 231, 1580-1584. Luo, L., Li, Y. and Kang, Y. (1990) Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells, Virology 179, 874-880. Mars, M., Zaguri, J.-F. and Fossati, I. (1990) Variable proteolytic cleavage of gag precursor expressed after infection of several cell lines with an HIV-2 gag-pol recombinant vaccinia virus. AIDS Res. Hum. Retroviruses 6. 271-273.
Moosmayer, D.. Reil, H.. Ausmeier. M., Scharf. J.-G., Hauser. H., Jentsch. K.D. and Hunsmann. G. (1991) Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-I gag and pol gene products in stably trnnsfected rodent cell lines. Virology 1X3. 215-224. Overton, H.A., Fujii, Y., Price, I.R. and Jones, I.M. (1989) The protease and gag gene products of the human immunodificiency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 170, lO7- 1 16. Schneider. J., Liike, W., Kirchhoff. F.. Jung, R., Jurkiewicz. E., Stahl-Hennig, C.. Nick. S., Klemm. E.. Jentsch. K.D. and Hunsmann. G. (1990) Isolation and characterization of HIV-2,,,, obtained from a patient with predominantly neurological defects. AIDS 4. 455-457. Shioda, T. and Shibuta, H. (1990) Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gqq gene of HIV. Virology 17.5. 139-14X. Smith, A.J.. Cho, M.-I., Hammarskjiild, M.-L. and Rekosh. D. (1990) Human immunodeficiency virus type 1 prS5 e’e and prlh0”““““’ expressed from a simian virus 40 late replacement vector arc efficiently processed and assembled into viruslike particles. J. Viral. 64, 2743-2750. Stahl-Hennig. C., HerchenrGder, 0.. Nick, S., Evers. M.. Stille-Siegener, M., Jentsch, K.D.. Kirchhoft. F.. Tolle, T., Gatesman. T.J.. Liike. W. and Hunsmann, G. (1990) Experimental infection of macaques with HIV-2,,,.. a novel HIV-2 isolate. AIDS 4, 61 l-617. Vzorov, A.N.. Bukrinsky. M.I.. Grigoriev. V.B., Tentsov, Y.Y. and Bukrinskaya, A.G. (1991) Highly immunogenic human immunodeficiency viruslike particles are produced by recombinant vaccinia virus-infected cells. AIDS Res. Hum. Retrovir. 7, 29-36.
VIRUS
197 B.V. All rights reserved
0168.1702/92/$05.00
00796
Morphogenesis
Gerald
of recombinant core particles
HIV-2 gag
Voss ‘, Frank Kirchhoff ‘, Sigrid Nick ‘, Dieter Moosmayer Hans Gelderblom ’ and Gerhard Hunsmann a
’ Deutsches
Primatenzentrum,
(Received
Abteilung Virolo@e und Immunologic, ’ Robert-Koch-lnstitut, Berlin, Germany
6 February
1992, revision
received
and accepted
GGttingen, Germany
18 March
‘, and
1992)
Summary
The gag-pol coding region of the HIV-2 nEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VW. These recombinant VV encoded either the whole gag-pal region or the gag gene including the proteasecoding region of the pol gene or the gag gene truncated at its 3’-end or only the pal gene. The HIV-2,,, gag precursor ~55, its mature cleavage products p24 and p17 as well as the pal reverse transcriptase CRT) p66 were detected in VV-infected CV-1 cells. The’p55 and two intermediate cleavage products p40 and ~35 were myristilated. Comparison to lysates of permanently HIV-2,,,-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3’-end of the gag gene coding for the p16 protein is essential for the
Correspondence to: Gerald Voss, Deutsches Kellnerweg 4, D-3400 GGttingen, Germany.
Primatenzentrum,
Abteilung
Virologie
und Immunologic,
198
formation of complete HIV-2 assembly of the viral core. HIV-2;
Gene
gag; Gene
particles
pol; Recombinant
and
that
the
VV; Particle
pal
proteins
support
the
assembly
Introduction The gag gene of human (HIV) and simian (SIV) immunodeficiency viruses codes for the major viral structural proteins. These proteins are processed from a precursor molecule (~55) into the core protein p24 and the inner coat protein ~17. The processing is mediated by the viral protease encoded at the 5’-terminus of the pol gene (Kramer et al., 1986; Debouck et al., 1987) in the gag-pal overlapping region. The expression of the pal gene occurs by frame shifting from the gag to the pal open reading frame (Jacks et al., 1988). The role of the viral protease for proper processing of the gag precursor has been investigated using recombinant proteins derived from various expression systems. Processing of the gag precursor by the viral protease was first reported in yeast cells by expression of the whole gag-pal region (Kramer et al., 1986). Coexpression of the viral protease and the gag precursor showed that the protease is responsible for the processing (Debouck et al., 1987; Overton et al., 1989). The processing of the gug precursor of HIV-l and HIV-2 expressed in mammalian cell lines depended on the host cell line (Flexner et al., 1988; Gowda et al.. 1989; Mars et al., 1990; Moosmayer et al., 1991). Recombinant viral particle structures were observed in mammalian cells infected with recombinant VV coding the HIV-l gag gene (Hu et al., 1990; Shioda and Shibuta, 1990) and with a gag-coding simian virus 40 replacement vector (Smith et al., 1990). These particles had an electron-dense ring-like shape of around 100 nm in diameter. Similar particles were produced in insect cells when infected with recombinant baculoviruses expressing the gug genes of HIV-l (Gheysen et al., 1989; Overton et al., 19891, HIV-2 (Luo et al., 19901 and SIVMA,. (Delchambre et al., 1989). Moreover, particles containing both HIV-l gag and enl’ proteins were observed after coinfection of mammalian cells with two recombinant VV containing the gag and the em’ gene (Haffar et al., 1990; Vzorov et al., 1991). In all cases the uncleaved gag precursor was able to form immature circular particles. Assembly to regular HIV-like core structures was only detected when processed p24 was present (Smith et al., 1990; Haffar et al., 1990; Karacostas et al., 1989). However, little was known about the regions of the gag and pal gene responsible for a proper particle assembly. In this report, we have examined the properties of recombinant gag and pal et al., 1990) expressed with recombinant VV. We proteins of HIV-2 nEN (Schneider showed the efficient expression of HIV-2 UEN gag and pal proteins with recombinant VV in CV-1 cells. In the presence of the protease-coding region of the pal gene, the gug precursor p55 was cleaved into the mature p24 and ~17 proteins.
199
Furthermore, an uncleaved gag p.59 precursor was processed after coexpression of the pal gene with respective VV. Using electron microscopy different states of virus maturation were detected. Early budding structures (VVgag >, circular immature particles (VVgag’) and mature HIV cores (VVgagpol) were formed by the recombinant gag proteins. The reported findings indicate that HIV-2 gag proteins are processed by the viral protease and that they are capable of self-assembly into immature retroviral particles and occasionally to mature core structures similar to HIV-l gag proteins. Furthermore, the gag p16 protein seems to be essential for complete budding.
Materials
and Methods
Cells and LTiruses in Molt 4 clone 8 The HIV-2 nEN isolate (Schneider et al., 19901 was propagated cells (Kikukawa et al., 1986) and used as a control in Western blot analyses of the recombinant gag and pol proteins. The HIV-2,,, genes for the construction of recombinant VV were derived from the proviral MK6 clone of HIV-2,,, (Kirchhoff et al., 1990a). Purified VV-DNA for in vivo recombination was made from VV wildtype (wt) strain Copenhagen. The recombination was carried out in CV-1 cells (Jensen et al., 1964) infected with the temperature-sensitive mutant VVts7 (Condit et al., 19831, which grows at 33°C and is inhibited at 39S”C. The selection for thymidine kinase (TK-)-defective VV with bromodeoxyuridine (BrDU) was performed in TK- mouse L-cells (LM (TK-I) (Kit et al., 1963). Recombinant rlaccinia [iruses The HIV-2,,, gag and pol genes were cloned into the VV insertion plasmid pTG186poly (Kieny et al., 1986). The plasmid contained the VV TK gene interrupted by the VV 7.5K promoter and a polylinker. Four different fragments of the HIV-2 nn,, genome (Kirchhoff et al., 1990b) (Fig. la) were inserted into the plasmid: (i) the gag gene truncated at its 3’-end overlapping the pol reading frame (gagg> (nucleotides 1107-25921, (ii) the gag gene including the protease-coding region of the pol gene (gag’) (1107-32121, (iii) the complete gag-pol region (gagpol) (1107-5758) and (iv> the pol gene (poll (2323-5758). The gag+ and gag-pol genes were subcloned into pTG186poly without further modification. Gag- was generated by excising most of the protease-coding region (downstream from 2592) from gag+ in pTG186gag+ and religation of the plasmid. This resulted in the extension of the reading frame into plasmid derived sequences coding for additional 85 amino acids. For subcloning in pTG186poly and expression of the pol gene, a Sal1 restriction site and an ATG start codon were incorporated at positions 2317 and 2323 of the HIV-2,,, sequence. Mutagenesis was essentially performed as described by Fritz et al. (1988), using the oligonucleotide 5’-
pal
\
:-’
;,
I
’
Fig. 1. Structure of HIV-2,,, gcr,e and &coding recombinant VV. (a) Four different regions of the ,qzg and pal open reading frames of the proviral HIV-2u,, clone were inserted into the VV insertion vector pTG186poly. The genetic structure of the resulting recombinant VV is shown. The crossed region of VVgug indicates the translated part of pTG186poly downstream the truncated gag gene and comprises 85 amino acids. The Sal1 * restriction site was generated by oligonucleotide-directed mutagenesis to generate VVpol (see Materials and Methods). (b) The resulting budding structures and particles after expression of the different recombinant VV are shown schematically.
GCAGCCCTGTCTTCATGGTCGACGGCACTGTCTTGC-3 ’ (mismatches are underlined) spanning bases 2303 to 2339 of the HIV-2nEN sequence. For in vivo recombination of the insertion plasmids 1 X 10” CV-1 cells were infected with 1 pfu per cell of VVts7. The VV were allowed to grow for 2 h at 33°C. The cells were washed once with serum-free Dulbecco’s modified Eagle medium (DMEM) and transfected with 250 ~1 of calcium-phosphate precipitate containing 200 ng insertion plasmid and 1 pug VVwt DNA. After 1 h, the precipitate was removed and the cells were washed twice with serum-free DMEM. Subsequently, the cells were incubated at 395°C in DMEM with 10% fetal calf serum (FCS) and after 48 h, the cultures were frozen and thawed twice. The supernatants were used to infect LM (TK-) cells and recombinant viruses were selected in DMEM containing 10% FCS, 0.5% agar noble (Difco, Detroit, USA), 0.5% LMP-agarose (BRL, Gaithersburg, USA) and 150 pg per ml BrDU. Well isolated plaques were recovered and recombinant VV were expanded on CV-1 cells. The plaque purification was repeated twice. The molecular structure of the recombinant VV was determined by restriction endonuclease digestion and Southern blot analysis. Detection of recombinant proteins To analyze the recombinant proteins by Western infected with 1 pfu per cell of recombinant VV. After
blotting, CV-1 cells were 24 h the cells were frozen
201
once and pelleted. The pellets were suspended in low salt extraction buffer (LSEB) (10 mM Tris, 0.14 M NaCl, 2 mM MgCl,, 1 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride and 0.5% (v/v) Nonidet P-40) and incubated for 15 min on ice. The cell debris were pelleted at 1000 X g and 25 pg protein from the supernatants were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in a 9-16% gradient gel. Proteins were transferred to nitrocellulose sheets at 350 mA overnight. The sheets were saturated with 3% dry milk powder in phosphate buffered saline (PBS), (120 mM NaCl, 17 mM Na,HPO,, 3 mM KH,PO,, pH 7.2) and an macaque antiserum (Stahl-Hennig et al., 1990) or monospecific HIV-2 a,,-specific rabbit antisera to HIV-l ~17, SIV,,, p24 or HIV-l RT were added in PBS with 0.2% NP-40 for 2 h at 37°C. After three washes, an alkaline phosphatase-conjugated goat anti-human or anti-rabbit antiserum (Dianova, Hamburg, Germany) was incubated for 1 h at 37°C. After four washes with TBS (20 mM Tris, 500 mM NaCl, pH 7.5) and a final wash with carbonate buffer (100 mM NaHCO,, 1 mM MgCl,, pH 9.8), the blots were developed with p-nitroblue tetrazoliumchloride (Biorad, Miinchen, Germany) and 5-bromo-4-chloro-3-indolyl-phosphatetoluidine (Sigma, Deisenhofen, Germany). (“H]myristic acid labeling 1 x 10h CV-1 cells were infected with 1 pfu of recombinant VV per cell and incubated for 16 h at 37°C in DMEM supplemented with 10% delipidated FCS. Then 50 PCi [“Hlmyristic acid (Amersham, Braunschweig, Germany) were added for 4 h and the cells were lysed in LSEB. The samples were centrifuged at 1000 x g, the supernatants were removed and the pellets were suspended in PBS. Supernatants and pellets were subjected to SDS-PAGE. The gel was fixed, amplified and dried. Labeled proteins were visualized by autoradiography. Electron microscopy Recombinant VV were used to infect CV-1 cells at 1 pfu per cell. After 24 h, when complete cytopathic effect was visible the cells were fixed in situ with 2.5% (v/v) glutardialdehyde in PBS. Cells were scraped off the plastic surface, sedimented at 275 x g and post fixed with 1% (w/v) 0~0,. After agar block enclosure cells were treated with 1% (v/v) tannic acid and processed further for Epon embedding using routine procedures (Gelderblom et al., 1987). Ultrathin sections were post-stained using Pb-citrate and examined at a Zeiss EM 10A electron microscope.
Results Recombinant HIV-2,,, in CV-1 cells
gag and pol proteins were expressed and efficiently processed
The recombinant HIV-2,,, gag and pol proteins infected with respective VV. Lysates of VV-infected
were expressed in CV-1 cells CV-1 cells were compared to
MI
IIIIIII
23456
i:.
366 -p59 -p55
-p24 ” pl7
Fig. 2. Western blot analysis of recombinant HIV-2 ,3,YAgcr,g XXI pal proteins expressed with recombinant VV. Lysates of CT-1 cells infected with VVgag(lane 2), VVgag’ (lane 3). VVgagpol (lane 4). VVpol (lane 5) or VVwt (lane 1) and HIV-I,,,:,-infected Molt-4 clone X cells (lane 6) were subjected to Western blot analysis using on HlV-2 ,,,,N-spccific macaque antiserum. Prestained protein markers (lane Mf had the following molecular wrights: 180, 116.84. 5X.4X.5and 36.5 kDa.
HIV-2,,,-infected Molt 4 clone 8 cells by Western blotting using an HIV-2,,,specific macaque serum (Fig. 2). Both, the recombinant VVgagpol and VVgag.+ included the protease-coding region of the pal gene. In CV-1 cells infected with these two recombinants. the gng precursor $5 was efficiently processed into the mature p24 and ~17. Both proteins exhibited the same elcctr~)phorctic mobility as the recombithe corresponding proteins in HIV-2 HE;N-infected cells. Furthermore, nant p24 and p17 as well as the pSS precursor were stained by respective monospecific rabbit antisera (data not shown). Radiolabeling of VV-infected CV- 1 cells with [‘Hjmyristic acid revealed that the recombinant pS5 gag precursor was myrist~lated (Fig. 3). Additionally, the expression of the recombinant g~~:ugproteins yielded some intermediate proteins, which did not occur in HIV-2,,,,-infected
203
Fig, 3. Myristilation of recombinant gag proteins. CV-1 cells were infected with VVwt (lane l), VVgag+ (lane 2), VVgag(lane 3) or VVgagpol (lane 4) and labeled with [“Hlmyristic acid (see Materials and Methods). After cell lysis the pellets were resuspended in PBS and separated electrophoretically. An autoradiography of the gel is shown.
cells. Two prominent bands at 40 and 35 kDa and an additional protein at 26 kDa were detected. Analysis with monospecific antisera revealed that the 26 kDa protein contained the p24 protein, while the other proteins comprised both, p24 and ~17 portions (data not shown). Like the ~5.5 precursor, the intermediate proteins at 40 and 35 kDa were myristilated (Fig. 3). The recombinant VVgagpol contained the complete gag and pol reading Western blot analysis showed that three additional proteins frames of HIV-2,,,.
204
were detectable in the respective cell lysates compared to VVgag+ (Fig. 2). One protein of 66 kDa was also present in HIV-2,,,infected cells and was stained by an RT-specific antiserum (data not shown). Two other proteins of 80 and 95 kDa were not found in HIV-2,,,-infected cells. The three proteins at 66, 80 and 95 kDa were also detected after infection of CV-1 cells with VVpol. Furthermore, six other recombinant proteins at 30, 53, 85, 90, 110 and 120 kDa were expressed in VVpol-infected cells. The protease-coding precwsor molecule
region of the pol gene is essential for processing
of the gag
To prove that the processing of the gag precursor p55 was mediated by the viral protease, a recombinant VV with a deletion at the 3’-end of the gag gene
I23 456-78910 I I I I I I I I I
Ml
I
I
P59 - p55 _
- p24
Fig. 4. Proteolytic cleavage of the pSY grog precursor by the viral protease. CV-I cells were infected with VVwt (lanes l-3) or VVpol (lanes 4-h) and after 4 h coinfected with 1 (lanes 1. 4), 5 (lanes 2, 5) or 20 (lanes 3, 6) pfu of VVgag-. After 24 h, cells were harvested and cytoplasmic lysates were subjected to SDS-polyacrylamide gel electrophoresis and subsequent Western blotting with a p24-specific rabbit antiserum. The lysates were compared to those of VVgag+ (lane 7). VVpol (lane 8) and VVgag(lane 9).infected CV-I cells and to HIV-2,,,:, infected Molt 4 clone X cells (lane 10).
20s
removing a part of the protease-coding region (VVgagg) was constructed. When this VVgag- was used to infect CV-1 cells, a gag precursor of 59 kDa was expressed (Fig. 21, which was myristilated like the recombinant p55 (Fig. 3). The high molecular weight of this precursor was due to expression of flanking regions of pTG186poly coding for additional 85 amino acids (Fig. la>. Although the precursor contained p24 and ~17, no processing into the mature proteins was observed. This shows that removal of the protease-coding region of the pol gene abolished the processing of the gag precursor.
Fig. 5. Retroviral were generated
particles formed by recombinant gag proteins. after infection with VVgagpol (e,f), VVgag+ represents 100 nm.
Electron micrographs of CV-1 cells (c,d) and VVgag(a,b). The bar
The recombinant protease of Wpol cleaL,es the gag precursor For further evaluation of the role of the viral protease, CV-1 cells were coinfected with VVgagand VVpol or VVwt. In cells coinfected with VVgag and VVwt the gag precursor was not processed, whereas coinfection with VVgag and VVpol resulted in production of small amounts of p24 (Fig. 4) depending on the quantity of ~59 precursor. This is a second evidence for the cleavage of the gag precursor by the viral protease. Formation of recombinant retroriral particles Finally, the recombinant gag proteins were investigated for their ability to self-assembly into retroviral particles and whether this assembly was depending on a proper processing of the gag precursor. Electron microscopy revealed that in CV-1 cells infected with VVgagtypical budding structures occurred. Virus formation did not proceed further and was apparently stopped at an early state in assembly (Fig. 5a,b). Expression of VVgag+ resulted in formation of particles with approximately 100 nm diameter, which were released in a normal way representing immature lentiviruses, except for the lack of envelope surface protein knobs. However, the released particles did not mature further (Fig. 5c,d). Fig. 5e,f show representative particles observed in cells infected with VVgagpol. Most of the cell-associated structures represent immature ~55 particles. Only a few mature virions were detected containing condensed mature tubular cores (Fig. 5f). A schematic drawing of the budding structures is shown in Fig. lb.
Discussion were expressed in CV-1 cells infected with The gag and pol genes of HIV-2,,, recombinant VV. The cleavage pattern of the gag precursor and the function of the viral protease was found to be similar to HIV-l. The recombinant proteins were identified with specific antisera and the gag precursor was shown to be myristilated. Deletion of the protease-coding region abolished processing of the gag precursor. The processing could be reconstituted by expression of the pol gene in VVgag--infected cells. Formation of budding structures occurred with unprocessed gag precursor and immature circular particles were observed after infection of CV-1 cells with VVgag+. Expression of VVgagpol yielded a few mature HIV cores. The expression of the gag and pol proteins with different recombinant VV yielded precursor and processed HIV-2 nEN proteins of the same size as they were detectable in HIV-2 a,,-infected Molt 4 clone 8 cells. In addition, monospecific antisera showed a reaction pattern with the recombinant proteins similar to that observed with HIV-2 nEN- infected cells. From these findings we conclude that the recombinant proteins produced by recombinant VV are identical to the naturally
207
occurring HIV-2 proteins. Beside these expected proteins, various additional gag intermediates and several &-related proteins were synthesized in the CV-1 cells infected with recombinant VV. These proteins were artificial products of the expression system. Similar differences to HIV-infected cells were found when HIV-l gag proteins were expressed with recombinant VV (Hu et al., 1990). The functional activity of the HIV protease also seems to depend on the cell type used for expression (Mars et al., 1990; Moosmayer et al., 1991) and on other factors, which remain unclear. For example, expression of the whole gag-pol region of HIV-l resulted in the production of mature gag proteins (Gheysen et al., 1989; Karacostas et al., 1989, Gowda et al., 1989), while other investigators did not observe a specific processing (Flexner et al., 1988; Shioda and Shibuta, 1990). The two HIV-2,,, gagpol and gag+ constructs showed efficient processing in several cell lines like Molt 4 clone 8, CEM, macaque B lymphoblastoid cells and BHK-21 cells. One exception was observed in LM (TK-) cells, where no processing was detected (data not shown). When the protease-coding region was deleted from the gag+ construct, the resulting gag precursor was not processed. Similar observations were reported for HIV-l gag precursors (Hu et al., 1990). Thus, absence of the viral protease is responsible for the lack of proteolytic cleavage. The coinfection of CV-1 cells with VVgagand VVpol showed a partial processing of the gag precursor ~59. This indicates that a functional protease was expressed by VVpol. Furthermore, this result reveals that the viral protease does not only cleave the adjacent gag precursor comprised in the large gagpol molecule as suggested by the results of Kramer et al. (1986), Gowda et al. (19891, Gheysen et al. (1989), Karacostas et al. (1989) and Hu et al. (1990), for HIV-l, but that it acts also on other gag precursors. This is consistent with observations of coexpression of the HIV-l protease and the gag precursor in E. coli (Debouck et al., 1987) and with baculoviruses in insect cells (Overton et al., 1989). Our electron microscopy observations suggest that the morphogenesis of HIV-2 core particles proceeds in several distinct steps. Other investigators have shown that the first step in immunodeficiency virus core assembly is the membrane anchorage of the gag precursor mediated by the myristic acid at its N-terminal end (Gheysen et al., 1989; Bryant and Ratner, 1990). Subsequently, budding structures are formed and particles with a circular electron-dense ring are released from the cell surface (Overton et al., 1989; Hu et al., 1990; Shioda and Shibuta, 1990; Gheysen et al., 1989; Luo et al., 1990; Delchambre et al., 1989). Our findings suggest that the C-terminal part of the gag precursor is also involved in the formation of complete particles. The gag precursor truncated in the pl6-coding region assembled only into incomplete budding structures. This phenomenon does not seem to be due to the lack of protease activity because the uncleaved gag precursor was repeatedly shown to form complete particles (Hu et al., 1990; Shioda and Shibuta, 1990; Gheysen et al., 1989; Luo et al., 1990; Delchambre et al., 1989). Deletion of the C-terminal portion of the SIV,,o gag precursor completely abolished particle formation and led to protrusions of the cell surface (Gheysen et al., 1989). Furthermore, mutations in the C-terminal p6 gag protein of HIV-l
20x
prevented the release of budded particles (Gottlinger et al., 1991). Hoshikawa et al. (1991) observed a complete lack of budding when the HIV-l p9 gag protein was deleted. These findings support our assumption of the requirement of p16 for complete particle formation, although it can not be excluded that the adjacent plasmid-derived portion of the HIV-2 nEN ~59 precursor may have prevented complete budding. The final step in particle assembly is the formation of the viral core consisting of ~24. A prerequisite for this process is the cleavage of p24 from the precursor by the protease (Gelderblom, 1991). In fact, mature HIV particles produced by recombinant expression systems were only observed when processed p24 was present (Overton et al., 1989; Haffar et al., 1990; Karacostas et al., 1989). The recombinant HIV-2 nEN proteins expressed by VVgag+including p24 did not assemble to mature particles. This was only observed when the whole gag-pol region was expressed. Immunoprecipitation of radiolabeled recombinant gag proteins revealed that higher amounts of cleaved p24 were produced by VVgagpol compared to VVgag+ (data not shown). Furthermore, the processing of the gug precursor from VVgagpol was not as efficient as in chronically HIV-2,,,-infected cells. Therefore the expression of the pal gene actively supports the formation of mature particles, possibly facilitating a more efficient processing of the gag precursor. Since HIV recombinant gag particles are immunogenic in rabbits (Luo et al., 1990; Vzorov et al., 1991) and additional viral proteins like the RT (Shioda and Shibuta, 1990) or the envelope glycoprotein (Haffar et al., 1990; Vzorov et al., 1991) can be included, these particles offer new perspectives for the development of vaccines against AIDS. In this context, it is of interest that the HIV-2,nN gag particles described in this study are derived from a human immunodeficiency virus able to infect macaques (Stahl-Hennig et al., 1990). Thus, the macaque model offers the opportunity to investigate the potential of HIV-2,,, gag particles as an AIDS vaccine.
Acknowledgements The authors would France) for supplying doctoral thesis of G.V.
like to thank Dr. M.-P. Kieny (Transgene, us with pTG186poly. This work contains
Strasbourg, parts of the
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