| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
2 Institute of Public Health, National Yang-Ming University, Taipei, Taiwan
3 AIDS Prevention and Research Center, National Yang-Ming University, Taipei, Taiwan
4 Department of Internal Medicine, Division of Infectious Disease, Taipei Veterans General Hospital, 201 Section 2 Shih-Pai Road, Taipei 11217, Taiwan
5 Department of Medical Research and Education, Taipei Veterans General Hospital, 201 Section 2 Shih-Pai Road, Taipei 11217, Taiwan
Correspondence
Chin-Tien Wang
chintien{at}ym.edu.tw
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Hunter, 1994). Pr55gag is transported to the plasma membrane where several thousand Pr55gag molecules assemble into virus particles that bud out from the cell membrane. During or after virus budding, Pr55gag is cleaved by the viral pol-encoded protease (PR) into four major proteins: matrix (MA; p17), capsid (CA; p24), nucleocapsid (NC; p7) and the C-terminal p6 (Erickson-Viitanen et al., 1989; Freed, 1998; Henderson et al., 1992; Kaplan et al., 1994; Leis et al., 1988; Mervis et al., 1988; Swanstrom & Wills, 1997). The gag and pol coding sequences partially overlap. A –1 ribosomal frameshift event occurs at a frequency of about 5 % during gag translation, resulting in Pol being translated as a Gag–Pol fusion protein (Jacks et al., 1988). Within the Gag–Pol, the p6 domain is truncated and replaced by a transframe domain referred to as p6* or p6pol (Partin et al., 1990). Proteolytic processing of Gag–Pol by the PR yields reverse transcriptase (RT), RNase H and integrase (IN) in addition to the Gag cleavage products. The PR-mediated virus maturation process is not required for virus assembly and budding, but is essential for viral infectivity (Gottlinger et al., 1989; Kohl et al., 1988; Peng et al., 1989).
How the PR is activated to mediate Gag particle maturation is still not completely understood. It is thought that dimerization of Gag–Pol is a prerequisite for PR activation (Navia & McKeever, 1990). The activated PR then cleaves itself out from Gag–Pol and functions as a homodimer to process Gag and Gag–Pol. An in vitro study has suggested that sequences flanking the PR domain may contribute to the process of PR activation (Louis et al., 1999; Pettit et al., 2003; Wondrak & Louis, 1996). Several other studies have also demonstrated that HIV-1 Gag–Pol molecules lacking the gag coding sequence or truncated in the RT domain are significantly defective in autoprocessing or in trans processing of Gag particles (Engelman et al., 1995; Liao & Wang, 2004; Quillent et al., 1996; Zybarth & Carter, 1995). Thus, sequences downstream or upstream of PR may potentially affect the PR activity, presumably via facilitating formation of a proper Gag–Pol dimer, which is thought to be required for activating the embedded PR.
The p6* domain is located directly N-terminal to the PR and separates NC from Pol. One previous study demonstrated that the removal of the p6* improves the proteolytic processing of Gag–Pol in vitro, suggesting an inhibitory effect of p6* on PR activation (Partin et al., 1991). In support of this notion, mutations preventing cleavage of p6* from the PR have been shown to markedly impair PR-mediated Gag processing (Chen et al., 2004; Tessmer & Krausslich, 1998; Zybarth et al., 1994). Additionally, synthetic p6* peptides have been reported to be able to suppress PR activity in vitro (Louis et al., 1998; Paulus et al., 1999). These results strongly suggests that the presence of p6* may interfere with the functioning of PR, and that p6* does not appear to make a positive contribution to the process of PR activation.
To investigate the role of the HIV-1 p6* domain in PR-mediated virus particle processing, deletion mutations in p6* were engineered by the two-megaprimer PCR extension method (Sambrook & Russell, 2001) using HIVgpt, which carries the SV40 ori and gpt (xanthine-guanine phosphoribosyltransferase) genes in the env region (Page et al., 1990), or a Pr160gag–pol-expression plasmid, GPfs (Chiu et al., 2002) as template. Primer sequences and detailed procedures for creating the mutations are available on request. Wild-type GPfs or each of the GPfs mutants (Fig. 1) was coexpressed with a Pr55gag expression plasmid, pGAG, in 293T cells. At 48 h post-transfection, culture medium from transfected 293T cells was filtered through 0.45 µm pore-size filters, followed by centrifugation through 2 ml 20 % sucrose in TSE [10 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA] plus 0.1 mM PMSF at 4 °C for 40 min at 274 000 g (SW41 rotor at 40 000 r.p.m.). The cells were rinsed with ice-cold PBS, pelleted, and were resuspended in 250 µl immunoprecipitation buffer plus 0.1 mM PMSF, and then subjected to microcentrifugation at 4 °C for 15 min at 13 700 g to remove cell debris. Supernatant and cell samples were prepared and subjected to Western immunoblot analysis as described previously (Wang et al., 1998). When cotransfected with pGAG plasmid at a DNA ratio of 1 : 10, both D2fs and D3fs exhibited a virus particle processing pattern similar to that of wild-type (wt) GPfs, with mature p24gag representing the major species of virus-associated Gag products (Fig. 2a, lanes 15 and 17). In contrast, D1fs produced a total amount of unprocessed and incompletely processed Gag slightly higher than that of wt (Fig. 2a, lanes 13 vs 11 and Fig. 2c, lanes 15 vs 13), suggesting that PR-mediated particle processing has been affected when most of the p6* codons were removed. However, virus particle production was markedly reduced or abolished when the amount of plasmid DNA of the wt GPfs or p6* deletion mutants used for cotransfection was equivalent to that of pGAG (Fig. 2a, lanes 10, 12, 14 and 16). This indicates that when both wt and mutant GPfs are overexpressed they can efficiently suppress virus budding, presumably due to premature cleavage of Gag precursors by Gag–Pol (Arrigo & Huffman, 1995; Burstein et al., 1991; Krausslich, 1991; Park & Morrow, 1991; Rose et al., 1995; Wang et al., 2000; Xiang et al., 1997).
|
|
; Zybarth et al., 1995) it was of interest to test the effects of a p6*-deleted mutation on PR-mediated virus processing in a Gag–Pol context containing a whole intact (p6gag/p6*Pol, p6gag/Pol) or deleted Gag domain [
GAG, (GAG+p6*); Fig. 1
]. Unlike GAG, which could still produce virus-associated p24gag (Fig. 2b, lane 12), (GAG+p6*) was severely defective in proteolytic processing of Pr55gag, as no mature p24gag was detected in either medium or cell lysates (Fig. 2b, lanes 6 and 13). Despite being cotransfected with a higher amount of plasmid DNA, (GAG+p6*) still could not produce p24/25gag; instead, a trace amount of p41gag was observed (Fig. 2b, lanes 3 and 10). This suggests that PR-mediated Gag processing was almost abrogated by the (GAG+p6*) mutation. In contrast, the p6gag/p6*Pol demonstrated a Gag particle processing profile similar to that of wt GPfs when cotransfected with pGAG at a DNA ratio of either 1 : 10 or 1 : 1 (Fig. 2c, lanes 16–17 vs 12–13). However, the p6gag/Pol was markedly defective in Gag particle processing, as substantial amounts of Gag proteins remained in the precursor or intermediate forms (Fig. 2c, lane 19). Overexpression of p6gag/Pol does not suppress virus particle production as efficiently as the GPfs or p6gag/p6*Pol (Fig. 2c, lanes 18 vs 12 and 16). These results suggests that the damage of p6*-deletion mutation to PR-mediated Gag processing is accentuated in the presence of additional mutations.
Although it is thought that the incorporation of HIV Gag–Pol into virus particles depends on interactions with the Pr55gag through its N-terminal Gag domain, it is unknown whether mutations in p6* can affect the incorporation of Gag–Pol into virus particles. To test whether p6* deletion mutations have any detrimental effect on Gag–Pol incorporation, which may consequently impair viral infectivity, aliquots of the culture medium used for infection were measured for virus-associated RT by Western blot. The results shown in Fig. 2(d) suggest that the p6* deletion mutations have no significant effect on the incorporation of Gag–Pol into virions, as D1fs, D2fs and D3fs all produced a level of virus-associated p66/51 RT comparable to that of wt GPfs (Fig. 2d, lanes 3–6). However, in addition to relatively higher levels of unprocessed and incompletely processed Gag, trace amounts of Pr160gag–pol precursors were readily detected in virions produced from cotransfections with D1fs (Fig. 2d, lane 4). This supports the proposal that the D1 mutation has impaired PR activity.
The results shown above suggest that our p6* deletion mutations in Gag–Pol have no major or only a modest effect on PR-mediated particle processing. Since the PR-mediated virus maturation process is a prerequisite for viral infectivity, immature or inappropriately processed virus particles ought to lose infectivity. We performed a single-cycle-infection assay to measure how the p6* deletion mutations affected viral infectivity. To do so, the wt GPfs or each of the mutant GPfs constructs was cotransfected with the pGAG plus a vesicular stomatitis virus glycoprotein expression vector, pHCMV-G (Yee et al., 1994). At 48–72 h, culture supernatants of transfected 293T cells were collected and the filtered supernatants were used to infect HeLa cells, which had been split and grown to 20 % confluence at the time of infection. Adsorption of virus was allowed to proceed at 37 °C in the presence of 4 µg Polybrene ml–1. Two days after infection, the cells were trypsinized and split 1 : 10 into 10 cm dishes containing selection medium (50 µg xanthine ml–1, 3 µg hypoxanthine ml–1, 4 µg thymidine ml–1, 10 µg glycine ml–1 and 150 µg glutamine ml–1) plus 25 µg mycophenolic acid (Gibco) ml–1 (Chen et al., 1997). Ten to 14 days later, colonies of drug-resistant cells were fixed and stained with 0.5 % methyl blue in 50 % methanol. Drug-resistant colonies were converted to titres (infectious units ml–1) and normalized to the corresponding virus-associated Gag protein level. As shown in Fig. 3(b), the infectivity of virions produced from cotransfections with D1fs or D2fs is markedly reduced, with viral infectivity at 20–50 % relative to that of wt GPfs. In contrast, D3fs could produce virus particles with a level of infectivity comparable to that of wt GPfs. HIVgptD3 showed an infectivity level of about 80 % compared with the wt (Fig. 3a), which agrees with previous reports that suggest deletions in this region do not significantly affect HIV-1 replication (Bleiber et al., 2004; Paulus et al., 2004). In the case of cotransfection with p6gag/p6*Pol, the infectivity of the released virions was only about 10 % relative to wt GPfs, although the virus particles were processed as well as those produced from the wt GPfs cotransfection (Fig. 2c, lanes 17 vs 13). These results suggest that a significant portion of the processed virions from cotransfections with Gag–Pol mutants were non-infectious. Functional interference in the post-assembly post-processing stage of virus replication by the p6gag embedded in the incorporated Gag–Pol may account in part for the reduced viral infectivity. The defect in viral particle processing is certainly able to impair viral infectivity. However, efficient PR-mediated virus processing is necessary but not sufficient for viral infectivity since several other factors may affect viral infectivity. For instance, the incorporated Gag–Pol mutants may have an impact on proper Gag assembly, and consequently interfere with virus replication. Additionally, tRNA incorporation (Mak et al., 1994) or the stability of genomic RNA dimer (Shehu-Xhilaga et al., 2001) may be influenced by the Gag–Pol mutants, resulting in reduced viral infectivity. This may possibly explain why D1fs, which is unable to process virus particles as well as the wt and contains a near wt level of RT, produced virions with infectivity reduced to only 20 % relative to that of the wt.
|
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bleiber, G., Peters, S., Martinez, R., Cmarko, D., Meylan, P. & Telenti, A. (2004). The central region of human immunodeficiency virus type 1 p6 protein (Gag residues S14–I31) is dispensable for the virus in vitro. J Gen Virol 85, 921–927.
Burstein, H., Bizub, D. & Skalka, A. M. (1991). Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers. J Virol 65, 6165–6172.
Chen, Y.-L., Ts'ai, P.-W., Yang, C.-C. & Wang, C.-T. (1997). Generation of infectious virus particles by transient co-expression of human immunodeficiency virus type 1 gag mutants. J Gen Virol 78, 2497–2501.[Abstract]
Chen, S.-W., Chiu, H.-C., Liao, W.-H., Wang, F. D., Chen, S.-S. & Wang, C.-T. (2004). The virus-associated human immunodeficiency virus type 1 Gag-Pol carrying an active protease domain in the matrix region is severely defective both in autoprocessing and in trans processing of gag particles. Virology 318, 534–541.[Medline]
Chiu, H.-C., Yao, S.-Y. & Wang, C.-T. (2002). Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag-Pol are not essential for incorporation of the Pr160gag-pol into virus particles. J Virol 76, 3221–3231.
Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A. & Craigie, R. (1995). Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 69, 2729–2736.[Abstract]
Erickson-Viitanen, S., Manfredi, J., Viitanen, P., Tribe, D. E., Tritch, R., Hutchison, C. A., III, Loeb, D. D. & Swanstrom, R. (1989). Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res Hum Retroviruses 5, 577–591.[Medline]
Freed, E. O. (1998). HIV Gag proteins: diverse functions in the virus life cycle. Virology 251, 1–15.[CrossRef][Medline]
Gottlinger, H. G., Sodroski, J. G. & Haseltine, W. A. (1989). Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus 1. Proc Natl Acad Sci U S A 86, 5781–5785.
Henderson, L. E., Bowers, M. A., Sowder, R. C., II Serabyn, S. A., Johnson, D. G., Bess, J. W., Jr, Arthur, L. O., Bryant, D. K. & Fenselau, C. (1992). Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processing, and complete amino acid sequences. J Virol 66, 1856–1865.
Hunter, E. (1994). Macromolecular interactions in the assembly of HIV and other retroviruses. Semin Virol 5, 71–83.
Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J. & Varmus, H. E. (1988). Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331, 280–283.[CrossRef][Medline]
Kaplan, A. H., Manchester, M. & Swanstorm, M. (1994). The activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency. J Virol 68, 6782–6786.
Kohl, N. E., Emini, E. A., Schleif, W. E., Davis, L. J., Heimbach, J. C., Dixon, R. A. F., Scolnick, E. M. & Sigal, I. S. (1988). Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A 85, 4686–4890.
Krausslich, H.-G. (1991). Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc Natl Acad Sci U S A 88, 3213–3217.
Leis, J., Baltimore, D., Bishop, J. B. & 8 other authors (1988). Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol 62, 1808–1809.
Liao, W.-H. & Wang, C.-T. (2004). Characterization of human immunodeficiency virus type 1 Pr160gag-pol mutants with truncations downstream of the protease domain. Virology 329, 180–188.[CrossRef][Medline]
Louis, J. M., Dyda, F., Nashed, N. T., Kimmel, A. R. & Davies, D. R. (1998). Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease. Biochemistry 37, 2105–2110.[CrossRef][Medline]
Louis, J. M., Clore, G. M. & Gronenborn, A. M. (1999). Autoprocessing of HIV-1 protease is tightly coupled to protein folding. Nat Struct Biol 6, 868–875.[CrossRef][Medline]
Mak, J., Jiang, M., Wainberg, M. A., Hammarskjöld, M. L., Rekosh, D. & Kleiman, L. (1994). Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol 68, 2065–2072.
Mervis, R. J., Ahmad, N., Lillehoj, E. P., Raum, M. G., Salazar, F. H. R., Chan, H. W. & Venkatesan, V. (1988). The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol 62, 3993–4002.
Navia, M. A. & McKeever, B. M. (1990). A role for the aspartyl protease from the human immunodeficiency type 1 (HIV-1) in the orchestration of virus assembly. Ann N Y Acad Sci 616, 73–85.[Abstract]
Page, K. A., Landau, N. R. & Littman, D. R. (1990). Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J Virol 64, 5270–5276.
Park, J. & Morrow, C. D. (1991). Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. J Virol 65, 5111–5117.
Partin, K., Krausslich, H. G., Ehrlich, L., Wimmer, E. & Carter, C. (1990). Mutational analysis of a native substrate of the human immunodeficiency virus type 1 proteinase. J Virol 64, 3938–3947.
Partin, K., Zybarth, G., Ehrlich, L., DeCrombrugghe, M., Wimmer, E. & Carter, C. (1991). Deletion of sequences upstream of the proteinase improves the proteolytic processing of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 88, 4776–4780.
Paulus, C., Hellebrand, S., Tessmer, U., Wolf, H., Krausslich, H.-G. & Wagner, R. (1999). Competitive inhibition of immunodeficiency virus type 1 protease by the Gag-Pol transframe protein. J Biol Chem 274, 21539–21543.
Paulus, C., Ludwig, C. & Wagner, R. (2004). Contribution of the Gag-Pol transframe domain p6* and its coding sequence to morphogenesis and replication of human immunodeficiency virus type 1. Virology 330, 271–283.[Medline]
Peng, C., Ho, B. K., Chang, T. W. & Chang, N. T. (1989). Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J Virol 63, 2550–2556.
Pettit, S. C., Gulnik, S., Everitt, L. & Kaplan, A. H. (2003). The dimer interfaces of protease and extra-protease domains influence the activation of protease and the stability of Gag-Pol cleavage. J Virol 77, 366–374.
Quillent, C., Borman, A. M., Paulous, S., Dauguet, C. & Clavel, F. (1996). Extensive regions of pol are required for efficient human immunodeficiency virus polyprotein processing and particle maturation. Virology 219, 29–36.[CrossRef][Medline]
Rose, J. R., Babe, L. M. & Craik, C. S. (1995). Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J Virol 69, 2751–2758.[Abstract]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shehu-Xhilaga, M., Crowe, S. M. & Mak, J. (2001). Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol 75, 1834–1841.
Swanstrom, R. & Wills, J. W. (1997). Synthesis, assembly, and processing of viral proteins. In Retroviruses. Edited by J. M. Coffin, S. H. Hughes & H. E. Varmus. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Tessmer, U. & Krausslich, H.-G. (1998). Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity. J Virol 72, 3459–3463.
Wang, C.-T., Lai, H.-Y. & Li, J.-J. (1998). Analysis of minimal human immunodeficiency virus type 1 gag coding sequences capable of virus-like particle assembly and release. J Virol 72, 7950–7959.
Wang, C.-T., Chou, Y.-C. & Chiang, C.-C. (2000). Assembly and processing of human immunodeficiency virus gag mutants containing a partial replacement of the matrix domain by the viral protease domain. J Virol 74, 3418–3422.
Wills, J. W. & Craven, R. C. (1991). Form, function, and use of retroviral gag proteins. AIDS 5, 639–654.[Medline]
Wondrak, E. M. & Louis, J. M. (1996). Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease. Biochemistry 35, 12957–12962.[CrossRef][Medline]
Xiang, Y., Ridky, T. W., Krishna, N. K. & Leis, J. (1997). Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation. J Virol 71, 2083–2091.[Abstract]
Yee, J. K., Friedmann, T. & Burns, J. C. (1994). Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43, 99–112.
Zybarth, G. & Carter, C. (1995). Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag-Pol influence PR autoprocessing. J Virol 69, 3878–3884.[Abstract]
Zybarth, G., Krausslich, H. G., Partin, K. & Carter, C. (1994). Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain. J Virol 68, 240–250.
Received 12 October 2005;
accepted 9 March 2006.
This article has been cited by other articles:
|
|
 |
|
  C. Ludwig, A. Leiherer, and R. Wagner Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation J. Virol., May 1, 2008; 82(9): 4573 - 4584. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
