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Role of the HIV gp120 Conserved Domain 1 in Processing and Viral Entry

2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês

10.1074/jbc.m806099200

1083-351X

Jizhen Wang, Jayita Sen, Lijun Rong, Michael Caffrey,

HIV/AIDS Research and Interventions

The importance of the N-terminal region of HIV gp120 conserved domain 1 (gp120-C1) to envelope function has been examined by alanine-scanning mutagenesis and subsequent characterization of the mutagenic effects on viral entry; envelope expression, processing, and incorporation; and gp120 association with gp41. With respect to the wild-type gp120, mutational effects on viral entry fall into two classes: functional, as defined by >20% entry with respect to wild type, and impaired, as defined by 20% entry with respect to wild type, and impaired, as defined by 20% entry with respect to wild type (E32A, K33A, I34A, V36A, T37A, K46A, E47A, T49A, T50A, and T51A); 2) impaired, as defined by <20% entry with respect to wild type (W35A, V38A, Y39A, Y40A, G41A, V42A, P43A, V44A, W45A, I52A, and F53A). With respect to the functional mutants, Val36 and Thr37 are highly conserved among HIV-1, HIV-2, and SIV; however, substitution of these residues with alanine is a relatively conservative change. With respect to the impaired mutations, Val38, Tyr40, Gly41, Pro43, Trp45, and Phe53 are highly conserved among HIV-1, HIV-2, and SIV. Interestingly, many of the largest effects to entry occur in the hydrophobic region encompassing Val38–Trp45. Envelope Expression and Processing—A Western blot analysis of wild-type and mutant envelope present in cell lysates was carried out to probe for mutational effects on expression and processing, as shown in Fig. 3. First note that the wild type shows the characteristic bands of gp160, gp120, and gp41, indicating that gp160 is expressed and processed by furin-like proteases to form mature gp120 and gp41. In the case of the mutants, the relatively high levels of gp160 present in cell lysates suggest that envelope expression has not been significantly affected by any of the substitutions. On the other hand, many of the mutations have reduced gp160 processing, as evidenced by reduced levels of gp120 and gp41. For example, W35A, V38A, Y39A, Y40A, G41A, V42A, P43A, W45A, I52A, and F53A exhibit undetectable or greatly reduced levels of gp120 and gp41 in the cell lysates, presumably due to a lack of processing by furin-like proteases. Not surprisingly, many of the mutants that display reduced gp160 processing also are impaired for viral entry (i.e. entry levels are 20% entry with respect to the wild type), and they exhibit the wild type pattern of gp160, gp120, and gp41 in cell extracts and virions. Consequently, this group is assigned a wild-type phenotype (Table 1). Impaired mutants P43A and W45A show reduced levels of virion incorporation and processing, and thus they exhibit a mixed phenotype. Impaired mutants W35A, V38A, Y39A, Y40A, G41A, V42A, and I52A exhibit a processing defect, as evidenced by decreased levels of gp120 and gp41 in cell lysates and virus (cf. Figs. 3 and 4 and Table 1). Note that the Y40A substitution is found in some HIV-1 subtype O strains, which suggests that other mutations compensate to allow processing in these strains. Impaired mutants V44A and F53A exhibit a defect in gp120-gp41 association. In the case of V44A, the alanine substitution occurs in the HIV-2 and SIV envelope (Fig. 1), which suggests that other mutations compensate to stabilize gp120-gp41 association.TABLE 1Summary of viral entry and the level of envelope detected in cell lysates and virus of the wild-type and alanine mutants of HIV gp120-C1 Entry levels are reported as the percentage of entry with respect to the wild-type virus. The reported levels are normalized to p24 levels observed in the Western blot of the virus (see "Materials and Methods"). The reported errors correspond to the S.D. of three separate experiments. Envelope levels are based on a densitometric analysis of Western blots. –, 140% (cf. supplemental Tables S1 and S2).MutantEntry (percentage of wild type)Cell gp160Cell gp120Cell gp41Virus gp160Virus gp120Virus gp41Phenotype%Wild type100 ± 12++++++++++++Wild typeE32A60 ± 12++++++++++++Wild typeK33A71 ± 15+++++++++Wild typeI34A108 ± 24+++++++++++Wild typeW35A0.4 ± 0.1+++––+––ProcessingV36A36 ± 6+++++++++Wild typeT37A40 ± 11++++++++++Wild typeV38A8.4 ± 4.0++++–+++–ProcessingY39A0.0 ± 0.1+++––++––ProcessingY40A0.0 ± 0.1+++––+++–ProcessingG41A16 ± 3+++–++++ProcessingV42A1.0 ± 0.2++––+––ProcessingP43A1.0 ± 0.4+++––+––Processing/IncorporationV44A14 ± 11+++++++–+Association/ProcessingW45A0.0 ± 0.1+++––+––Processing/IncorporationK46A83 ± 5++++++++++Wild typeE47A76 ± 7+++++++++Wild typeT49A82 ± 21+++++++++Wild typeT50A53 ± 16++++++++++Wild typeT51A60 ± 21+++++++++Wild typeI52A10 ± 4++––++––ProcessingF53A3.2 ± 0.9++––++–+Association/Processing Open table in a new tab Previous Studies of gp120-C1—A number of previous studies have characterized the effects of mutations in HIV gp120-C1 on viral function, and thus it is of interest to compare the conclusions of these studies in light of the present work. For example, Ivey-Hoyle et al. (14Ivey-Hoyle M. Clark R. Rosenberg M. J. Virol. 1991; 65: 2682-2685Crossref PubMed Google Scholar) observed that deletion of the 31 aminoterminal residues of gp120 (residues 31–57 of gp120-C1) (Fig. 1) did not disrupt envelope processing or CD4 binding but did result in dissociation of gp120 from gp41, which suggested that the gp120-C1 domain directly interacts with gp41. The V44A and F53A mutants of the present study, which are found within this region, also disrupt gp120 association with gp41 and thus support this notion. In another study, Helseth et al. (15Helseth E. Olshevsky U. Furman C. Sodroski J. J. Virol. 1991; 65: 2119-2123Crossref PubMed Google Scholar) found that single-site mutants V36L and Y40D were processed and bound CD4 but resulted in dissociation of gp120 from gp41. In the present study, the analogous mutant V36A exhibited a wild-type phenotype, with the difference presumably due to the nature of the substituting group (e.g. the leucine substitution is more bulky than valine and may result in steric hindrance). Moreover, the Y40A mutant of the present study disrupted envelope processing, with the difference probably due to the smaller and more hydrophobic nature of alanine with respect to aspartate. In addition, Helseth et al. (15Helseth E. Olshevsky U. Furman C. Sodroski J. J. Virol. 1991; 65: 2119-2123Crossref PubMed Google Scholar) found that mutant W45S reduced processing and gp120 association with gp41. In agreement, the analogous W45A mutant of the present study also reduced processing; however, the effects on gp120-gp41 association could not be assayed due to the complete absence of processing in this mutant. Finally, we note that the present study represents the first single site mutations to Glu32, Lys33, Ile34, Trp35, Thr37, Val38, Tyr39, Gly41, Val42, Pro43, Val44, Lys46, Glu47, Thr49, Thr50, Thr51, Ile52, and Phe53. Of the novel mutations, mutants W35A, V38A, Y49A, V42A, and I52A exhibit significantly impaired function due to processing defects, mutant P43A exhibits impaired function due to defects in processing and virion incorporation, and mutants V44A and F53A exhibit impaired function due to disruption of the gp120-gp41 association. Taken together, the gp120-C1 region plays important roles in envelope processing and stabilization of the gp120-gp41 interaction. Interactions of gp120-C1 in gp120/gp41—As mentioned in the Introduction, large regions of the gp120-C1 and gp120-C5 domains are missing from the available structures of the gp120 core (10Chen B. Vogan E. Gong H. Skehel J. Wiley D. Harrison S. Nature. 2005; 433: 834-841Crossref PubMed Scopus (463) Google Scholar, 11Kwong P. Wyatt R. Robinson T. Sweet R. Sodroski J. Hendrickson W. Nature. 1998; 393: 648-659Crossref PubMed Scopus (2491) Google Scholar, 12Huang C. Tang M. Zhang M. Majeed S. Montabana E. Stanfield R. Dimitrov D. Korber B. Sodroski J. Wilson I. Science. 2005; 310: 1025-1028Crossref PubMed Scopus (642) Google Scholar), and thus mutagenesis and immunological studies play important roles in the characterization of these domains, which are highly conserved between HIV-1, HIV-2, and SIV (9Douglas N. Munro G. Daniels R. J. Mol. Biol. 1997; 273: 122-149Crossref PubMed Scopus (41) Google Scholar). However, it is important to note that single site mutational effects can be due to direct interactions or interactions propagated to more distant sites. Nonetheless, we would like to consider intermolecular interactions between gp120-C1 and gp120-C5 with the gp41 disulfide loop in processed gp160 (Fig. 5). The gp120-C1 domain is shown with the secondary structure predicted by Hansen et al. (30Hansen J. Lund O. Nielsen J. Brunak S. Hansen J. Proteins. 1996; 25: 1-11Crossref PubMed Scopus (32) Google Scholar). The gp120-C5 structure is taken from Guilhaudis et al. (13Guilhaudis L. Jacobs A. Caffrey M. Eur. J. Biochem. 2002; 269: 4860-4867Crossref PubMed Scopus (22) Google Scholar), and the gp41 disulfide loop structure is taken from Refs. 25Caffrey M. Biochim. Biophys. Acta. 2001; 1536: 116-122Crossref PubMed Scopus (65) Google Scholar and 31Caffrey M. Cai M. Kaufman J. Stahl S. Wingfield P. Gronenborn A. Clore G. EMBO J. 1998; 17: 4572-4584Crossref PubMed Scopus (368) Google Scholar. Note that the gp120-C5 structure has the caveat that it was determined in the presence of 40% trifluoroethanol, a co-solvent that stabilizes helical structure, and in the absence of other gp120 and gp41 domains (13Guilhaudis L. Jacobs A. Caffrey M. Eur. J. Biochem. 2002; 269: 4860-4867Crossref PubMed Scopus (22) Google Scholar). The gp41 disulfide loop has the caveat that it is based on the postfusion form of gp41 (i.e. the six-helix bundle); however, we are not aware of any evidence for a large structural change of the disulfide loop during HIV entry. In Fig. 5, residues implicated by mutagenesis studies in forming intermolecular interactions in processed gp160 are shown in green. The interaction between Val44 and Phe53 of gp120-C1 and gp41 is based on the present work, in which single site mutations resulted in disruption of the gp120-gp41 interaction. The intermolecular interactions of Ile491 of gp120-C5 and Trp596 and Ser618 of the gp41 disulfide loop are based on previous mutagenesis studies, suggested by dissociation of gp120 from gp41 (19Jacobs A. Sen J. Rong L. Caffrey M. J. Biol. Chem. 2005; 280: 27284-27288Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Sen J. Jacobs A. Caffrey M. Biochemistry. 2008; 47: 7788-7795Crossref PubMed Scopus (28) Google Scholar). The interaction between A501 of gp120-C5 and Thr605 of the gp41 disulfide loop is based on the SOS mutant of Binley et al. (16Binley J. Sanders R. Clas B. Schuelke N. Master A. Guo Y. Kajumo F. Anselma J. Maddon P. Olson W. Moore J. J. Virol. 2000; 74: 627-643Crossref PubMed Scopus (449) Google Scholar), in which the double mutant A501C/T605C was shown to form a nonnative disulfide bond, and thus a direct interaction between these residues was implied. As noted above, Val36, Tyr40, and Trp45 have also been previously implicated in forming intermolecular interactions with gp41, based on gp120 dissociation from gp41 (15Helseth E. Olshevsky U. Furman C. Sodroski J. J. Virol. 1991; 65: 2119-2123Crossref PubMed Google Scholar). Importantly, immunological studies have also suggested interactions between the gp120-C1, gp120-C5, and the gp41 disulfide loop (17Moore J. Willey R. Lewis G. Robinson J. Sodroski J. J. Virol. 1994; 68: 6836-6847Crossref PubMed Google Scholar, 18Wyatt R. Desjardin E. Olshevsky U. Nixon C. Binley J. Olshevsky V. Sodroski J. J. Virol. 1997; 71: 9722-9731Crossref PubMed Google Scholar), thereby supporting the schematic model of processed gp160. Finally, note that the topology of the gp120 core structures imply that gp120-C1 and -C5 are in close proximity (10Chen B. Vogan E. Gong H. Skehel J. Wiley D. Harrison S. Nature. 2005; 433: 834-841Crossref PubMed Scopus (463) Google Scholar, 11Kwong P. Wyatt R. Robinson T. Sweet R. Sodroski J. Hendrickson W. Nature. 1998; 393: 648-659Crossref PubMed Scopus (2491) Google Scholar, 12Huang C. Tang M. Zhang M. Majeed S. Montabana E. Stanfield R. Dimitrov D. Korber B. Sodroski J. Wilson I. Science. 2005; 310: 1025-1028Crossref PubMed Scopus (642) Google Scholar). Interactions of gp120-C1 in gp160—It is next of interest to examine potential long range intramolecular interactions between gp120-C1, gp120-C5, and the gp41 disulfide loop in unprocessed gp160. In Fig. 5, residues implicated in forming long range interactions with the furin recognition site in unprocessed gp160 are shown in blue. Specifically, the interaction between Trp35, Val38, Tyr39, Tyr40, Gly41, Val42, Pro43, Trp45, and Ile52 of gp120-C1 and the furin recognition site of gp120-C5 is based on the data presented herein, in which alanine substitutions resulted in severely decreased envelope processing. The interaction between Leu593 of the gp41 disulfide loop and the furin recognition site is based on a previous mutagenesis study (19Jacobs A. Sen J. Rong L. Caffrey M. J. Biol. Chem. 2005; 280: 27284-27288Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The notion that the gp41 disulfide bond interacts with gp120-C5 in gp160 is also supported by the mutagenesis study of Sen et al. (20Sen J. Jacobs A. Jiang H. Rong L. Caffrey M. Protein Sci. 2007; 16: 1236-1241Crossref PubMed Scopus (18) Google Scholar), who showed that the disulfide bond within the loop was important to gp160 processing. Interestingly, the present study suggests that long range interactions between gp120-C1 and gp120-C5, which encompasses the furin recognition site, occur in unprocessed gp160. Indeed, the impaired entry of many of the gp120-C1 mutants was due to a lack of processing, underscoring the importance of gp120-C1 residues to proper presentation of the gp120-C5 furin recognition site, a site that is ∼450 residues away (cf. Fig. 1). Ivey-Hoyle et al. (14Ivey-Hoyle M. Clark R. Rosenberg M. J. Virol. 1991; 65: 2682-2685Crossref PubMed Google Scholar) have shown that deletion of the N-terminal 31 residues of gp120 (residues 31–57 of gp120-C1) (Fig. 1) does not significantly reduce envelope processing, which may appear to be in disagreement with the results presented herein. It is tempting to speculate that deletion of gp120-C1, in contrast to single site mutations, may remove residues with the potential to reduce access to the furin recognition site, thereby allowing envelope processing. In summary, the extreme sensitivity of gp120-C1 to alanine substitutions suggests that this region is an attractive and novel target for future drug discovery efforts. For example, compounds that bind to gp120-C1 may be expected to disrupt gp120 function and hence HIV entry, by disrupting either gp160 processing or gp120 association with gp41. Reagents pNL4-3.Luc.R-E-, pCONBgp160opt, U87.CD4.CCR5, and HIV-1 gp41 Hybridoma (Chessie 8) were obtained through the National Institutes of Health AIDS Research and Reference Program. We thank Caitlin Ondracek for aid in the preparation of the gp120-C1 mutants. Download .pdf (.05 MB) Help with pdf files