Vol. 66, No. 3
JOURNAL OF VIROLOGY, Mar. 1992, p. 1799-1803
0022-538X/92/031799-05$02.00/0 Copyright ©) 1992, American Society for Microbiology
Mutational Analysis of Conserved N-Linked Glycosylation Sites of Human Immunodeficiency Virus Type 1 gp4l WOAN-RUOH LEE, XIAO-FANG
YU, WAN-JR SYU,t MAX ESSEX, AND TUN-HOU LEE*
Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts 02115 Received 10 July 1991/Accepted 4 December 1991
Amino acid substitutions were introduced into four conserved N-linked glycosylation sites of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein, gp4l, to alter the canonical N-linked glycosylation sequences. One altered site produced a severe impairment of viral infectivity, which raises the possibility that N-linked sugars at this site may have an important role in the human immunodeficiency virus type 1 life cycle. The molecule that provides the membrane anchor for the human immunodeficiency virus type 1 (HIV-1) external envelope protein, gpl20 (1), is the envelope transmembrane protein, gp4l (22). The coding sequence of gp4l is unusually long compared with the transmembrane protein of retroviruses outside the subfamily of lentiviruses (14, 17, 18, 21, 23). The extra coding sequence of gp4l is located distal to a characteristic hydrophobic membrane anchor domain and is believed to be on the cytoplasmic side of the membrane (2). Except for this unique feature, gp41 has several structural features, within the region proximal to the hydrophobic membrane anchor domain, in common with the envelope transmembrane proteins of a divergent family of animal retroviruses (5, 21). These common features, from the N terminus to the C terminus of gp4l, include the following: a hydrophobic stretch of amino acid residues, a region rich in threonine and serine residues, a string of amino acid residues with a high probability of forming an a-helix structure, two or three vicinal cysteine residues, and consensus N-linked glycosylation sites which are followed by the hydrophobic membrane anchor domain. The functional significance of some of the conserved structural features of gp4l was explored in several previous studies. For instance, mutations introduced to the hydrophobic sequences in the N terminus of gp4l were found to greatly reduce the syncytium-forming abilities of the mutant viruses (8). Mutagenesis through insertion of in-frame linkers to the region rich in threonine and serine residues was found to disrupt the association between gp4l and gpl20 (9). In addition, substituting the two highly conserved vicinal cysteine residues of gp4l with other amino acid residues was found to affect the processing of the gpl60 precursor, which suggests that the conformation dictated by the disulfide bond formed by these vicinal cysteine residues is important for the maturation of the envelope protein (20). In the coding sequence for gp41 of most HIV-1 isolates with known nucleotide sequences, there are four consensus N-linked glycosylation sites located within a region flanked by two highly conserved vicinal cysteine residues and a hydrophobic membrane anchor domain (15). This high degree of structural conservation is intriguing in light of the sequence heterogeneity found in the envelope genes of * Corresponding author. t Present address: Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, Taiwan.
various HIV-1 isolates (15). In the present study, we used an HIV-1 molecular clone, HXB2 (4), as a working model to address the question of whether alteration of these highly conserved N-linked glycosylation sites of gp4l affects HIV-1 infectivity. As shown in Fig. 1, five potential N-linked glycosylation sites are present within the region flanked by the two highly conserved vicinal cysteine residues and the hydrophobic membrane anchor domain of HXB2. Among these five sites, only the first four sites are shared by most HIV-1 isolates (15). Five N-linked glycosylation site mutants of HXB2, designated 611, 616, 624, 637, and 674 (Fig. 1), which had the asparagine residue of the canonical N-linked glycosylation sequence replaced by a noncanonical residue, were constructed to study whether these conserved N-linked sequences were critical for HIV-1 infectivity. These mutants were generated by the oligonucleotide-directed mutagenesis of Kunkel et al. (10). The template used was a 2.7-kb SalI-BamHI fragment of HXB2, which contains most of the envelope-coding sequence of HXB2. The mutagenic oligonucleotides used to generate these mutants are shown in Table 1. As shown in Fig. 1, the asparagine residue of the canonical Asn-X-Ser/Thr sequence was replaced by a histidine residue. The SalI-BamHI fragments containing the desired mutations were verified by DNA sequencing (19), and the M13 replicative forms containing the desired mutations were used to replace the corresponding SalI-BamHI fragment of the wild-type virus. Western blot (immunoblot) analyses were first carried out to examine whether alterations introduced into these conserved N-linked sites affected the expression of the envelope gene-encoded gp160 and gpl20 (3). For this purpose, each proviral DNA of the wild-type and mutant viruses was transfected into COS-7 cells by the DEAE-dextran method (25). Cell lysates were prepared 48 h posttransfection and analyzed with a reference serum from an HIV-1-seropositive patient. As shown in Fig. 1, gp160 and gp120 were detected in the cells transfected with each of the five N-linked glycosylation site mutants and the wild-type virus. The specificities of gpl60 and gp120 were further indicated by the absence of these two species in the mock-transfected COS-7 cells and by the reactivities of these two species to a hyperimmune sheep antiserum to gpl20 (AIDS Research and Reference Reagent Program no. 288; data not shown). These results indicated that the expression of the envelope precursor, gpl60, and its subsequent cleavage to gpl20 were not 1799
1800
NOTES
J. VIROL.
gpl2Q
gp41
Mock
WT 611 616 624 637 674 518
V,gm:
-f~c
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NS N NT '.1 1* H HA HA HV
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+ t-
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-MS *flg~ an
-
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-
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-
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-
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_
_
ft4-o46
92.5
-
- 69
46
A
-
_
30
H
_ w4 __w4w
_
_
7
WT 611 616 624 637 674
Mock
WT 611 613 616 618
a
518
WT 624 637 639 674
-
9v:
925 69
-gpl2 gp4l
-
FIG. 1. (Top) Locations of five potential N-linked glycosylation sites within the extracellular domain of HXB2 gp4l. C, conserved cysteine residue; closed circles, the five potential N-linked glycosylation sites (Asn-X-Ser/Thr); hatched box, the hydrophobic membrane anchor sequence; N, S, T, H, A, and V, asparagine, serine, threonine, histidine, alanine, and valine, respectively; numbers, amino acid positions according to the numbering system of Ratner et al. (17); "Slight," mutants with subtle impairment of viral infectivity. (Bottom) Expression of gpl60 and gp120 in COS-7 cells transfected by the wild-type (WT) or mutant viruses. Cell lysates prepared from COS-7 transfectants were reacted with a reference HIV-1-positive human serum. The positions of gpl60 and gpl20 are
A
Mock WT
drastically affected by the alteration of the canonical N-linked glycosylation sequences at all four highly conserved N-linked glycosylation sites and a fifth nonconserved N-linked glycosylation site within the extracellular domain of gp4l. Lysates prepared from virions collected from the culture supernatants of the COS-7 cultures transfected by the wildtype virus or the mutant viruses were also analyzed by Western blot with a pool of HIV-1-positive sera showing positive reactivity to gp4l. As shown in Fig. 2, alterations introduced at the four conserved N-linked glycosylation sites caused all four mutated gp4l species to migrate faster than the wild-type gp4l. In contrast, an identical substitution of an asparagine residue with a histidine residue at the nonconserved site 674 had no discernible effect on the migration pattern of this mutated gp4l. The specificity of gp4l was indicated by its reactivity to a rabbit anti-gp4l TABLE 1. Synthetic oligonucleotides used for the construction of HIV-1 mutants
611
Mutagenic oligonucleotide (5' to 3')a
GCAGTGGGAATAGaAGCTTaGTTCCTTGGGTTCTTG GTGCCTTGGcATGCTAGTTG
613
CTTGGAATGCTgcTTGGAGTAATAA
616
TTGGAGTcATAAATCTCTGGAA
624
GGAGTAATAAAgCTtTGGAACAGAT CAGATCTGGcATCACACGACC
637
AGAAATTAACcATTACACAAGCT
639
AGAGAAATTAACAATTACgtcAGCTTAATACACTCCTT
674
GAATTGGTTTcACATAACAAATTG
618
a
Lowercase letters indicate mutation sites.
637
-
30
674
92 5 69
vow"mU
p53 -
518
611 616 624 518
p64 -
indicated.
Mutant
46
^^
gp4l
_m 4_ am
*
-
p34p24 -
_
e
3
_
"mm se
s
40
- 46
_
_ _e4gM4 q.p _b ___
-30
FIG. 2. Detection of wild-type and mutant gp4l in cell-free virions by Western blot analysis. Viral lysates were prepared from cell-free supernatants of the wild-type (WT)- or mutant (611, 616, 624, 637, 674, and 518)-transfected COS-7 cells. (Top) Viral lysates were reacted with a pool of four HIV-1-positive human serum samples. The positions of pol gene-encoded p64, p53, and p34, gag gene-encoded p24, and gp4l are indicated. (Middle) Viral lysates were reacted with a rabbit anti-gp4l serum. (Bottom) Viral lysates were treated with PNGase-F (Genzyme Corp., Cambridge, Mass.) according to the manufacturer's suggested procedures. The treated lysates were analyzed by Western blot with the same pool of HIV-1-positive human serum mentioned in the legend to Fig. 1. gp4l*, position of the deglycosylated gp4l.
serum (kindly provided by S. Alexander of Cambridge Biotech Corp., Worcester, Mass.) and by the absence of gp4l in the virions prepared from mock-transfected COS-7 cells or from mutant 518-transfected COS-7 cells (Fig. 2). Mutant 518 had a stop codon inserted at the seventh amino acid residue of gp4l (Table 1) and was not expected to have gp4l in its virions. One possible interpretation of these findings is that N-linked sugars are present at all four highly conserved potential N-linked glycosylation sites. This interpretation is particularly attractive for mutants 611 and 616 in light of our finding that after virions were treated with
peptide-N4-(N-acetyl-p-glucosaminyl)asparagine amidase, the deglycosylated gp4l of mutants 611 and 616 comigrated with the deglycosylated gp4l of the wild-type virus (Fig. 2).
VOL. 66, 1992
NOTES
1801
Luu o Mock * WT Aa 611 * 613
o Mock * WT a 624
-
Q
100
a
s-100-
CI.cc
E
3
6 10 13 17 20 24 DAYS POST-INFECTION
DAYS POST-INFECTION
100, 80
o Mock * WT A 637
80
* 639 60
60
Er
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I-
c
20
20
0L
O0
DAYS POST-INFECTION
DAYS POST-INFECTION
Mock
-* WT a
°
674
/
80
40-
___
3
6
10
13
17
20
24
DAYS POST-INFECTION FIG. 3. In vitro growth properties of the wild-type and N-linked glycosylation mutants in CD4-positive SupTl cells. The procedure for the RT assay was described previously (26). "Mock," mock-infected cultures; WT, wild type.
To study whether HIV-1 infectivity was affected by alterations introduced to these five N-linked glycosylation sites of gp4l, cell-free virions were collected 48 h posttransfection from COS-7 cultures transfected with mutant and wild-type virus. After adjustments for the level of reverse transcriptase (RT) activity were made, equal amounts of mutant and wild-type virus were used to infect a CD4-positive cell line, SupTl. The RT activities in the supernatants of the infected SupTI cultures were monitored every 3 or 4 days. Among the five N-linked glycosylation site mutants, the growth kinetics of mutant 637 was substantially delayed compared with that of the wild-type virus (Fig. 3). While the RT activity was detected in the wild-type-virus-infected cultures 10 days postinfection, the RT activity was not detected in mutant 637-infected cultures until 27 days postinfection (Fig. 3). Slight delays in growth kinetics as monitored by the RT activity were observed in cultures infected by mutant 611 or mutant 616, with mutant 611 showing more delay than mutant 616. No delay in growth kinetics was observed for mutant 624 or mutant 674. These results suggest that some of the conserved N-linked glycosylation sites within the extracellular domain of gp4l may play roles in HIV-1 infectivity. The infectivities of three other N-linked glycosylation site mutants, designated 613, 618, and 639 (Fig. 1), which had the
threonine residue of the canonical Asn-X-Ser/Thr by a noncanonical alanine or valine studied. These third-site mutants were constructed to study whether the observed effects on viral infectivity for mutants 611, 616, and 637 were due to amino acid substitutions introduced at the first site of the canonical Asn-X-Ser/Thr sequence per se rather than to the loss of N-linked glycosylation sites. If a third-site mutant has a phenotype similar to that of the wild-type virus, it is highly unlikely that the observed effect on viral infectivity of the corresponding first-site mutant can be attributed to the loss of an N-linked glycosylation site. Both gpl60 and gpl20 were detected in COS-7 cells transfected by mutant 613 (Fig. 1). No delay in growth kinetics was observed for this mutant compared with the wild-type virus (Fig. 3). Thus, the observed effect on viral infectivity for mutant 611 was likely due to the substitution of a histidine residue for an aspara-
serine
or
sequence replaced residue, were also
gine residue.
For mutant 618, no RT activity in cultures infected by this mutant was detected throughout the entire follow-up period (Fig. 3). The relative amounts of gpl60 and gpl20 in the
wild-type-virus-transfected COS-7 cells were compared by Western blot analysis with those in the mutant 618-transfected COS-7 cells. As shown in Fig. 1, the amount of gpl20
..vsa:es
C
MW 200
J. VIROL.
NOTES
1802
Mlock W- 618
MocK
E9'8
--
gp
92 5-
69--
ie
*w
120
4
a
b
46
FIG. 4. Radioimmunoprecipitation analysis of gpl20 present in cell lysates and virus-free culture supernatants of mutant 618transfected COS-7 cultures. Cell lysates and virus-depleted culture supernatants were derived from COS-7 cultures metabolically labelled with [35S]cysteine and transfected with no DNA (mock), wild-type DNA (WT), or mutant 618 proviral DNA and reacted with a reference HIV-1-positive human serum. The positions of gp160 and gp120 are indicated. MW, molecular weight standards.
in the mutant 618-transfected COS-7 cells was disproportionate to that of gpl60 (Fig. 1). It appears that the phenotype of mutant 618 can be explained by the excessive secretion of gp120, possibly as the result of dissociation from gp4l. As shown in Fig. 4, gpl60 and gpl20 in the wild-type-virustransfected COS-7 cells were detected by radioimmunoprecipitation analysis (11). In COS-7 cells transfected by mutant
618, however, gpl60 was
more readily detected than gpl20 and most of the gpl20 was detected in the culture supernatant. The loss of infectivity by mutant 618 could be attributed to excessive secretion of gpl20. It is unlikely that the loss of infectivity is caused by the loss of N-linked sugars, since a similar phenotype was not observed with mutant 616. These findings leave the question of whether N-linked sugars have a role in the slight impairment of infectivity by mutant 616 unresolved. It should be noted, however, that the amino acid residue altered by mutant 618 is outside the regions previously reported to be critical for the association between gpl20 and gp4l (9). Severe impairment of infectivity was observed for mutant 639. The level of RT activity detected in cultures infected by mutant 639 was not significantly above that of the mockinfected cultures until 20 days postinfection, which was a delay of 10 days compared with the wild-type virus (Fig. 3). Both gpl60 and gpl20 were detected in the mutant 639transfected COS-7 cells (Fig. 1). The mutated gp4l species detected in the virions also migrated faster than the wildtype virus (data not shown), which is compatible with the suggestion that N-linked sugars may be present in this conserved N-linked glycosylation site. One possible interpretation of the finding that both mutant 637 and mutant 639 have severe impairment of infectivity is that N-linked sugars at this particular N-linked glycosylation site may have important roles in HIV-1 infectivity. However, the alternative interpretation that the impairment of viral infectivity was caused by amino acid substitutions per se rather than by the loss of N-linked sugars cannot be ruled out. The N-linked sugars of HIV-1 have been proposed as potential targets for antiviral therapeutic agents (6, 7, 12, 13, 24). In fact, some inhibitors of the N-linked glycosylation pathway were previously shown to reduce HIV-1 infectivity (13, 16). However, it remains unclear which HIV-1 glyco-
proteins are targeted by these inhibitors. Our results raise the possibility that glycosylation inhibitors aimed at the gp4l N-linked glycosylation site represented by mutants 637 and 639 may have an antiviral effect. We thank Y. Chow and Z. Matsuda for helpful discussions; A. Wolf, B. Du, and M. F. McLane for technical assistance; and E. Conway for editorial assistance. This work was supported by Public Health Service grants CA39805 and HL-33774 from the National Institutes of Health and contract DAMD 17-90-C-0151 from the U.S. Army. W.-R. Lee was supported by training grant D43TW00004 from the Fogarty International Center, National Institutes of Health. REFERENCES 1. Allan, J., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A. Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091-1094. 2. Berman, P. W., W. M. Nunes, and 0. K. Haffar. 1988. Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines. J. Virol. 62:3135-3142. 3. Chou, M. J., T. H. Lee, A. Hatzakis, T. Mandalaki, M. F. McLane, and M. Essex. 1988. Antibody responses in early human immunodeficiency virus type I infection in hemophiliacs. J. Infect. Dis. 157:805-811. 4. Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Nature (London) 316:262-265. 5. Gallaher, W. R., J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro. 1989. A general model for the transmembrane proteins of HIV and other retroviruses. AIDS Res. Hum. Retroviruses 5:431-440. 6. Gruters, R. A., J. J. Neetjes, M. Tersmette, R. E. de Goede, A. TuIp, H. G. Huisman, F. Miedema, and H. L. Ploegh. 1987. Interference with HIV-induced syncytiurn formation and viral infectivity by inhibitors of trimming glucosidase. Nature (London) 330:74-77. 7. Karpas, A., G. W. Fleet, R. A. Dwek, S. Petursson, S. K. Namgoong, N. G. Ramsden, G. S. Jacob, and T. W. Rademacher. 1988. Amino sugar derivatives as potential anti-human immunodeficiency virus agents. Proc. Natl. Acad. Sci. USA 85:9229-9233. 8. Kowalski, M., L. Bergeron, T. Dorfman, W. Haseltine, and J. Sodroski. 1991. Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein. J. Virol. 65'281-291. 9. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351-1355. 10. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382. 11. Lee, T. H., J. E. Coligan, T. Homma, M. F. McLane, N. Tachibana, and M. Essex. 1984. Human T-cell leukemia virusassociated membrane antigens (HTLV-MA): identity of the major antigens recognized following virus infection. Proc. Natl. Acad. Sci. USA 81:3856-3860. 12. Matthews, T. J., K. J. Weinhold, H. K. Lyerly, A. J. Langlois, H. Wigzell, and D. P. Bolognesi. 1987. Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gpl20 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. Proc. Natl. Acad. Sci. USA 84:54245428. 13. Montefiori, D. C., W. E. Robinson, Jr., and W. M. Mitchell. 1988. Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 85:9248-9252. 14. Muesing, M. A., D. H. Smith, C. D. Cabradilla, C. V. Benton, L. A. Lasky, and D. J. Capon. 1985. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus.
NOTES
VOL. 66, 1992
Nature (London) 313:450-458. 15. Myers, G., A. B. Rabson, S. F. Josephs, T. F. Smith, and F. Wong-Staal. 1989. Human retroviruses and AIDS 1989: a compilation and analysis for nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N. Mex. 16. Pal, R., V. S. Kalyanaraman, G. M. Hoke, and M. G. Sarngadharan. 1989. Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin. Intervirology 30:27-35. 17. Ratner, L., W. Haseltine, R. Patarca, K. J. Livak, B. Starcich, S. F. Josephs, E. R. Doran, J. A. Rafalski, E. A. Whitehorn, K. Baumeister, L. Ivanoff, S. R. Petteway, Jr., M. L. Pearson, J. A. Lautenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C. Gallo, and F. Wong-Staal. 1985. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature (London) 313:277-284. 18. Sanchez Pescador, R., M. D. Power, P. J. Barr, K. S. Steimer, M. M. Stempien, S. L. Brown Shimer, W. W. Gee, A. Renard, A. Randolph, and J. A. Levy. 1985. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227:484-492. 19. Sanger, F., and A. R. Coulson. 1975. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94:441-445. 20. Syu, W. J., W. R. Lee, B. Du, Q. C. Yu, M. Essex, and T. H. Lee. 1991. Role of conserved gp4l cysteine residues in the
21.
22.
23. 24.
25. 26.
1803
processing of human immunodeficiency virus envelope precursor and viral infectivity. J. Virol. 65:6349-6352. VanBeveren, C., J. Coffin, and S. Hughes. 1985. Nucleotide sequences completed with functional and structural analysis, appendix B, p. 567-1148. In R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.), RNA tumor viruses, 2nd ed., vol. 2. Supplements and indexes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Veronese, F. D., A. L. DeVico, T. D. Copeland, S. Oroszlan, R. C. Gallo, and M. G. Sarngadharan. 1985. Characterization of gp4l as the transmembrane protein coded by the HTLV-III/ LAV envelope gene. Science 229:1402-1405. Wain Hobson, S., P. Sonigo, 0. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17. Walker, B. D., M. Kowalski, W. C. Goh, K. Kozarsky, M. Krieger, C. Rosen, L. Rohrschneider, W. A. Haseltine, and J. Sodroski. 1987. Inhibition of human immunodeficiency virus syncytium formation and virus replication by castanospermine. Proc. Natl. Acad. Sci. USA 84:8120-8124. Yu, X. F., S. Ito, M. Essex, and T. H. Lee. 1988. A naturally immunogenic virion-associated protein specific for HIV-2 and SIV. Nature (London) 335:262-265. Yu, X. F., M. Matsuda, M. Essex, and T. H. Lee. 1990. Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein. J. Virol. 64:5688-5693.
JOURNAL OF VIROLOGY, Mar. 1992, p. 1799-1803
0022-538X/92/031799-05$02.00/0 Copyright ©) 1992, American Society for Microbiology
Mutational Analysis of Conserved N-Linked Glycosylation Sites of Human Immunodeficiency Virus Type 1 gp4l WOAN-RUOH LEE, XIAO-FANG
YU, WAN-JR SYU,t MAX ESSEX, AND TUN-HOU LEE*
Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts 02115 Received 10 July 1991/Accepted 4 December 1991
Amino acid substitutions were introduced into four conserved N-linked glycosylation sites of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein, gp4l, to alter the canonical N-linked glycosylation sequences. One altered site produced a severe impairment of viral infectivity, which raises the possibility that N-linked sugars at this site may have an important role in the human immunodeficiency virus type 1 life cycle. The molecule that provides the membrane anchor for the human immunodeficiency virus type 1 (HIV-1) external envelope protein, gpl20 (1), is the envelope transmembrane protein, gp4l (22). The coding sequence of gp4l is unusually long compared with the transmembrane protein of retroviruses outside the subfamily of lentiviruses (14, 17, 18, 21, 23). The extra coding sequence of gp4l is located distal to a characteristic hydrophobic membrane anchor domain and is believed to be on the cytoplasmic side of the membrane (2). Except for this unique feature, gp41 has several structural features, within the region proximal to the hydrophobic membrane anchor domain, in common with the envelope transmembrane proteins of a divergent family of animal retroviruses (5, 21). These common features, from the N terminus to the C terminus of gp4l, include the following: a hydrophobic stretch of amino acid residues, a region rich in threonine and serine residues, a string of amino acid residues with a high probability of forming an a-helix structure, two or three vicinal cysteine residues, and consensus N-linked glycosylation sites which are followed by the hydrophobic membrane anchor domain. The functional significance of some of the conserved structural features of gp4l was explored in several previous studies. For instance, mutations introduced to the hydrophobic sequences in the N terminus of gp4l were found to greatly reduce the syncytium-forming abilities of the mutant viruses (8). Mutagenesis through insertion of in-frame linkers to the region rich in threonine and serine residues was found to disrupt the association between gp4l and gpl20 (9). In addition, substituting the two highly conserved vicinal cysteine residues of gp4l with other amino acid residues was found to affect the processing of the gpl60 precursor, which suggests that the conformation dictated by the disulfide bond formed by these vicinal cysteine residues is important for the maturation of the envelope protein (20). In the coding sequence for gp41 of most HIV-1 isolates with known nucleotide sequences, there are four consensus N-linked glycosylation sites located within a region flanked by two highly conserved vicinal cysteine residues and a hydrophobic membrane anchor domain (15). This high degree of structural conservation is intriguing in light of the sequence heterogeneity found in the envelope genes of * Corresponding author. t Present address: Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, Taiwan.
various HIV-1 isolates (15). In the present study, we used an HIV-1 molecular clone, HXB2 (4), as a working model to address the question of whether alteration of these highly conserved N-linked glycosylation sites of gp4l affects HIV-1 infectivity. As shown in Fig. 1, five potential N-linked glycosylation sites are present within the region flanked by the two highly conserved vicinal cysteine residues and the hydrophobic membrane anchor domain of HXB2. Among these five sites, only the first four sites are shared by most HIV-1 isolates (15). Five N-linked glycosylation site mutants of HXB2, designated 611, 616, 624, 637, and 674 (Fig. 1), which had the asparagine residue of the canonical N-linked glycosylation sequence replaced by a noncanonical residue, were constructed to study whether these conserved N-linked sequences were critical for HIV-1 infectivity. These mutants were generated by the oligonucleotide-directed mutagenesis of Kunkel et al. (10). The template used was a 2.7-kb SalI-BamHI fragment of HXB2, which contains most of the envelope-coding sequence of HXB2. The mutagenic oligonucleotides used to generate these mutants are shown in Table 1. As shown in Fig. 1, the asparagine residue of the canonical Asn-X-Ser/Thr sequence was replaced by a histidine residue. The SalI-BamHI fragments containing the desired mutations were verified by DNA sequencing (19), and the M13 replicative forms containing the desired mutations were used to replace the corresponding SalI-BamHI fragment of the wild-type virus. Western blot (immunoblot) analyses were first carried out to examine whether alterations introduced into these conserved N-linked sites affected the expression of the envelope gene-encoded gp160 and gpl20 (3). For this purpose, each proviral DNA of the wild-type and mutant viruses was transfected into COS-7 cells by the DEAE-dextran method (25). Cell lysates were prepared 48 h posttransfection and analyzed with a reference serum from an HIV-1-seropositive patient. As shown in Fig. 1, gp160 and gp120 were detected in the cells transfected with each of the five N-linked glycosylation site mutants and the wild-type virus. The specificities of gpl60 and gp120 were further indicated by the absence of these two species in the mock-transfected COS-7 cells and by the reactivities of these two species to a hyperimmune sheep antiserum to gpl20 (AIDS Research and Reference Reagent Program no. 288; data not shown). These results indicated that the expression of the envelope precursor, gpl60, and its subsequent cleavage to gpl20 were not 1799
1800
NOTES
J. VIROL.
gpl2Q
gp41
Mock
WT 611 616 624 637 674 518
V,gm:
-f~c
N0I'
NS
NS N NT '.1 1* H HA HA HV
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RcdtwICd 111fCCti\iti
+ t-
+ --
I) Z
p64 p53 gp4l
t
t
_
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N
-MS *flg~ an
-
S
wa
_u
c
- am - am am - ur
-
!--
p34
-
_
p24
-
_w
-m
_
_
ft4-o46
92.5
-
- 69
46
A
-
_
30
H
_ w4 __w4w
_
_
7
WT 611 616 624 637 674
Mock
WT 611 613 616 618
a
518
WT 624 637 639 674
-
9v:
925 69
-gpl2 gp4l
-
FIG. 1. (Top) Locations of five potential N-linked glycosylation sites within the extracellular domain of HXB2 gp4l. C, conserved cysteine residue; closed circles, the five potential N-linked glycosylation sites (Asn-X-Ser/Thr); hatched box, the hydrophobic membrane anchor sequence; N, S, T, H, A, and V, asparagine, serine, threonine, histidine, alanine, and valine, respectively; numbers, amino acid positions according to the numbering system of Ratner et al. (17); "Slight," mutants with subtle impairment of viral infectivity. (Bottom) Expression of gpl60 and gp120 in COS-7 cells transfected by the wild-type (WT) or mutant viruses. Cell lysates prepared from COS-7 transfectants were reacted with a reference HIV-1-positive human serum. The positions of gpl60 and gpl20 are
A
Mock WT
drastically affected by the alteration of the canonical N-linked glycosylation sequences at all four highly conserved N-linked glycosylation sites and a fifth nonconserved N-linked glycosylation site within the extracellular domain of gp4l. Lysates prepared from virions collected from the culture supernatants of the COS-7 cultures transfected by the wildtype virus or the mutant viruses were also analyzed by Western blot with a pool of HIV-1-positive sera showing positive reactivity to gp4l. As shown in Fig. 2, alterations introduced at the four conserved N-linked glycosylation sites caused all four mutated gp4l species to migrate faster than the wild-type gp4l. In contrast, an identical substitution of an asparagine residue with a histidine residue at the nonconserved site 674 had no discernible effect on the migration pattern of this mutated gp4l. The specificity of gp4l was indicated by its reactivity to a rabbit anti-gp4l TABLE 1. Synthetic oligonucleotides used for the construction of HIV-1 mutants
611
Mutagenic oligonucleotide (5' to 3')a
GCAGTGGGAATAGaAGCTTaGTTCCTTGGGTTCTTG GTGCCTTGGcATGCTAGTTG
613
CTTGGAATGCTgcTTGGAGTAATAA
616
TTGGAGTcATAAATCTCTGGAA
624
GGAGTAATAAAgCTtTGGAACAGAT CAGATCTGGcATCACACGACC
637
AGAAATTAACcATTACACAAGCT
639
AGAGAAATTAACAATTACgtcAGCTTAATACACTCCTT
674
GAATTGGTTTcACATAACAAATTG
618
a
Lowercase letters indicate mutation sites.
637
-
30
674
92 5 69
vow"mU
p53 -
518
611 616 624 518
p64 -
indicated.
Mutant
46
^^
gp4l
_m 4_ am
*
-
p34p24 -
_
e
3
_
"mm se
s
40
- 46
_
_ _e4gM4 q.p _b ___
-30
FIG. 2. Detection of wild-type and mutant gp4l in cell-free virions by Western blot analysis. Viral lysates were prepared from cell-free supernatants of the wild-type (WT)- or mutant (611, 616, 624, 637, 674, and 518)-transfected COS-7 cells. (Top) Viral lysates were reacted with a pool of four HIV-1-positive human serum samples. The positions of pol gene-encoded p64, p53, and p34, gag gene-encoded p24, and gp4l are indicated. (Middle) Viral lysates were reacted with a rabbit anti-gp4l serum. (Bottom) Viral lysates were treated with PNGase-F (Genzyme Corp., Cambridge, Mass.) according to the manufacturer's suggested procedures. The treated lysates were analyzed by Western blot with the same pool of HIV-1-positive human serum mentioned in the legend to Fig. 1. gp4l*, position of the deglycosylated gp4l.
serum (kindly provided by S. Alexander of Cambridge Biotech Corp., Worcester, Mass.) and by the absence of gp4l in the virions prepared from mock-transfected COS-7 cells or from mutant 518-transfected COS-7 cells (Fig. 2). Mutant 518 had a stop codon inserted at the seventh amino acid residue of gp4l (Table 1) and was not expected to have gp4l in its virions. One possible interpretation of these findings is that N-linked sugars are present at all four highly conserved potential N-linked glycosylation sites. This interpretation is particularly attractive for mutants 611 and 616 in light of our finding that after virions were treated with
peptide-N4-(N-acetyl-p-glucosaminyl)asparagine amidase, the deglycosylated gp4l of mutants 611 and 616 comigrated with the deglycosylated gp4l of the wild-type virus (Fig. 2).
VOL. 66, 1992
NOTES
1801
Luu o Mock * WT Aa 611 * 613
o Mock * WT a 624
-
Q
100
a
s-100-
CI.cc
E
3
6 10 13 17 20 24 DAYS POST-INFECTION
DAYS POST-INFECTION
100, 80
o Mock * WT A 637
80
* 639 60
60
Er
40 2-
I-
c
20
20
0L
O0
DAYS POST-INFECTION
DAYS POST-INFECTION
Mock
-* WT a
°
674
/
80
40-
___
3
6
10
13
17
20
24
DAYS POST-INFECTION FIG. 3. In vitro growth properties of the wild-type and N-linked glycosylation mutants in CD4-positive SupTl cells. The procedure for the RT assay was described previously (26). "Mock," mock-infected cultures; WT, wild type.
To study whether HIV-1 infectivity was affected by alterations introduced to these five N-linked glycosylation sites of gp4l, cell-free virions were collected 48 h posttransfection from COS-7 cultures transfected with mutant and wild-type virus. After adjustments for the level of reverse transcriptase (RT) activity were made, equal amounts of mutant and wild-type virus were used to infect a CD4-positive cell line, SupTl. The RT activities in the supernatants of the infected SupTI cultures were monitored every 3 or 4 days. Among the five N-linked glycosylation site mutants, the growth kinetics of mutant 637 was substantially delayed compared with that of the wild-type virus (Fig. 3). While the RT activity was detected in the wild-type-virus-infected cultures 10 days postinfection, the RT activity was not detected in mutant 637-infected cultures until 27 days postinfection (Fig. 3). Slight delays in growth kinetics as monitored by the RT activity were observed in cultures infected by mutant 611 or mutant 616, with mutant 611 showing more delay than mutant 616. No delay in growth kinetics was observed for mutant 624 or mutant 674. These results suggest that some of the conserved N-linked glycosylation sites within the extracellular domain of gp4l may play roles in HIV-1 infectivity. The infectivities of three other N-linked glycosylation site mutants, designated 613, 618, and 639 (Fig. 1), which had the
threonine residue of the canonical Asn-X-Ser/Thr by a noncanonical alanine or valine studied. These third-site mutants were constructed to study whether the observed effects on viral infectivity for mutants 611, 616, and 637 were due to amino acid substitutions introduced at the first site of the canonical Asn-X-Ser/Thr sequence per se rather than to the loss of N-linked glycosylation sites. If a third-site mutant has a phenotype similar to that of the wild-type virus, it is highly unlikely that the observed effect on viral infectivity of the corresponding first-site mutant can be attributed to the loss of an N-linked glycosylation site. Both gpl60 and gpl20 were detected in COS-7 cells transfected by mutant 613 (Fig. 1). No delay in growth kinetics was observed for this mutant compared with the wild-type virus (Fig. 3). Thus, the observed effect on viral infectivity for mutant 611 was likely due to the substitution of a histidine residue for an aspara-
serine
or
sequence replaced residue, were also
gine residue.
For mutant 618, no RT activity in cultures infected by this mutant was detected throughout the entire follow-up period (Fig. 3). The relative amounts of gpl60 and gpl20 in the
wild-type-virus-transfected COS-7 cells were compared by Western blot analysis with those in the mutant 618-transfected COS-7 cells. As shown in Fig. 1, the amount of gpl20
..vsa:es
C
MW 200
J. VIROL.
NOTES
1802
Mlock W- 618
MocK
E9'8
--
gp
92 5-
69--
ie
*w
120
4
a
b
46
FIG. 4. Radioimmunoprecipitation analysis of gpl20 present in cell lysates and virus-free culture supernatants of mutant 618transfected COS-7 cultures. Cell lysates and virus-depleted culture supernatants were derived from COS-7 cultures metabolically labelled with [35S]cysteine and transfected with no DNA (mock), wild-type DNA (WT), or mutant 618 proviral DNA and reacted with a reference HIV-1-positive human serum. The positions of gp160 and gp120 are indicated. MW, molecular weight standards.
in the mutant 618-transfected COS-7 cells was disproportionate to that of gpl60 (Fig. 1). It appears that the phenotype of mutant 618 can be explained by the excessive secretion of gp120, possibly as the result of dissociation from gp4l. As shown in Fig. 4, gpl60 and gpl20 in the wild-type-virustransfected COS-7 cells were detected by radioimmunoprecipitation analysis (11). In COS-7 cells transfected by mutant
618, however, gpl60 was
more readily detected than gpl20 and most of the gpl20 was detected in the culture supernatant. The loss of infectivity by mutant 618 could be attributed to excessive secretion of gpl20. It is unlikely that the loss of infectivity is caused by the loss of N-linked sugars, since a similar phenotype was not observed with mutant 616. These findings leave the question of whether N-linked sugars have a role in the slight impairment of infectivity by mutant 616 unresolved. It should be noted, however, that the amino acid residue altered by mutant 618 is outside the regions previously reported to be critical for the association between gpl20 and gp4l (9). Severe impairment of infectivity was observed for mutant 639. The level of RT activity detected in cultures infected by mutant 639 was not significantly above that of the mockinfected cultures until 20 days postinfection, which was a delay of 10 days compared with the wild-type virus (Fig. 3). Both gpl60 and gpl20 were detected in the mutant 639transfected COS-7 cells (Fig. 1). The mutated gp4l species detected in the virions also migrated faster than the wildtype virus (data not shown), which is compatible with the suggestion that N-linked sugars may be present in this conserved N-linked glycosylation site. One possible interpretation of the finding that both mutant 637 and mutant 639 have severe impairment of infectivity is that N-linked sugars at this particular N-linked glycosylation site may have important roles in HIV-1 infectivity. However, the alternative interpretation that the impairment of viral infectivity was caused by amino acid substitutions per se rather than by the loss of N-linked sugars cannot be ruled out. The N-linked sugars of HIV-1 have been proposed as potential targets for antiviral therapeutic agents (6, 7, 12, 13, 24). In fact, some inhibitors of the N-linked glycosylation pathway were previously shown to reduce HIV-1 infectivity (13, 16). However, it remains unclear which HIV-1 glyco-
proteins are targeted by these inhibitors. Our results raise the possibility that glycosylation inhibitors aimed at the gp4l N-linked glycosylation site represented by mutants 637 and 639 may have an antiviral effect. We thank Y. Chow and Z. Matsuda for helpful discussions; A. Wolf, B. Du, and M. F. McLane for technical assistance; and E. Conway for editorial assistance. This work was supported by Public Health Service grants CA39805 and HL-33774 from the National Institutes of Health and contract DAMD 17-90-C-0151 from the U.S. Army. W.-R. Lee was supported by training grant D43TW00004 from the Fogarty International Center, National Institutes of Health. REFERENCES 1. Allan, J., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A. Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091-1094. 2. Berman, P. W., W. M. Nunes, and 0. K. Haffar. 1988. Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines. J. Virol. 62:3135-3142. 3. Chou, M. J., T. H. Lee, A. Hatzakis, T. Mandalaki, M. F. McLane, and M. Essex. 1988. Antibody responses in early human immunodeficiency virus type I infection in hemophiliacs. J. Infect. Dis. 157:805-811. 4. Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Nature (London) 316:262-265. 5. Gallaher, W. R., J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro. 1989. A general model for the transmembrane proteins of HIV and other retroviruses. AIDS Res. Hum. Retroviruses 5:431-440. 6. Gruters, R. A., J. J. Neetjes, M. Tersmette, R. E. de Goede, A. TuIp, H. G. Huisman, F. Miedema, and H. L. Ploegh. 1987. Interference with HIV-induced syncytiurn formation and viral infectivity by inhibitors of trimming glucosidase. Nature (London) 330:74-77. 7. Karpas, A., G. W. Fleet, R. A. Dwek, S. Petursson, S. K. Namgoong, N. G. Ramsden, G. S. Jacob, and T. W. Rademacher. 1988. Amino sugar derivatives as potential anti-human immunodeficiency virus agents. Proc. Natl. Acad. Sci. USA 85:9229-9233. 8. Kowalski, M., L. Bergeron, T. Dorfman, W. Haseltine, and J. Sodroski. 1991. Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein. J. Virol. 65'281-291. 9. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351-1355. 10. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382. 11. Lee, T. H., J. E. Coligan, T. Homma, M. F. McLane, N. Tachibana, and M. Essex. 1984. Human T-cell leukemia virusassociated membrane antigens (HTLV-MA): identity of the major antigens recognized following virus infection. Proc. Natl. Acad. Sci. USA 81:3856-3860. 12. Matthews, T. J., K. J. Weinhold, H. K. Lyerly, A. J. Langlois, H. Wigzell, and D. P. Bolognesi. 1987. Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gpl20 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. Proc. Natl. Acad. Sci. USA 84:54245428. 13. Montefiori, D. C., W. E. Robinson, Jr., and W. M. Mitchell. 1988. Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 85:9248-9252. 14. Muesing, M. A., D. H. Smith, C. D. Cabradilla, C. V. Benton, L. A. Lasky, and D. J. Capon. 1985. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus.
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1803
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