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Mutational Analyses and Natural Variability of the gp41 Ectodomain

 

Rogier W. Sanders1, Bette Korber2, Min Lu3, Ben Berkhout1, and John P. Moore4

1 Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; r.w.sanders@amc.uva.nl

2 Theoretical Biology and Biophysics, MS K710, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

3 Department of Biochemistry and 4Department of Microbiology and Immunology, Weill Medical College, Cornell University, New York, New York 10021

 

The HIV-1 envelope glycoproteins mediate viral attachment and release of the viral core in susceptible target cells. A single gp160 precursor protein is processed intracellularly to yield the native form of the envelope complex, consisting of three gp120 and three gp41 molecules associated through non-covalent interactions. Upon receptor and co-receptor binding to the surface subunit gp120, conformational changes within the envelope glycoprotein complex enable the insertion of the hydrophobic fusion peptide of the transmembrane subunit gp41 into the target membrane. Subsequent rearrangements within gp41 allow fusion of viral and cellular membranes. These late structural alterations are targeted by the entry inhibitor T-20 (for reviews see 13, 20, 21, 24, 46, 75).

A considerable body of mutagenesis data on structure-function relationships within the HIV-1 gp41 ectodomain (gp41e) has been published over the years. The value of this data-set has been increased considerably by the determination of the structure of the gp41e core, allowing some of the mutational effects to be interpreted and at least partially understood (9, 12, 38, 41, 68, 71). The native, pre-fusion structure of gp41e in the trimeric gp120-gp41 complex on the virion surface prior to receptor engagement is not known, however, and the various transitional structures of gp41 during the virus-cell fusion process are still ill-defined. Consequently, the structural and functional consequences of many amino acid substitutions in gp41e remain unclear.

Here, we have summarized the results of published mutagenesis studies on gp41e (see the accompanying table). The HXB2 reference strain has been used as a basis for numbering individual amino acid residues (Figure 1). This information should facilitate the research of those who study the HIV-1 envelope glycoproteins as fusogens or vaccine antigens. In general, we have tabulated only data for single mutants, but several publications contain information on the effects of multiple amino acid substitutions (25, 43, 44, 49, 56, 57, 62). The table does not include information on every naturally occurring gp41e sequence variant, as the variation is extensive. However, a summary of natural variability in clades B and C is presented in Figure 2. Also, the last two columns in the table present the entropy scores for gp41e positions that have a defined impact on Env function, for both the B clade and the C clade. Not surprisingly, positions identified through mutational analysis as those where substitutions can abrogate key functions, also tend to be highly conserved among the natural variants. The clearest example is provided by positions where substitutions essentially eliminate cell-cell fusion (i.e., where fusion efficiencies in syncytium assays or reporter gene assays have been reduced to less than 3% of the wild-type value). Sites at which substitutions can abrogate cell-cell fusion tended to be more invariant among 123 B clade sequences (26/44, 59%), compared to those sites where amino acid changes did not dramatically reduce fusion (11/39, 28%, Fisher's exact test p = 0.004). Some unusual gp41e variants found in neutralization-resistant isolates are also included in the table, as are variants that arise in response to selection pressure, both in vitro and in vivo, from the entry inhibitor T-20, which targets gp41e.

The precision with which the available data could be analyzed was sometimes limited because different viral clones, isolates and assays were used to obtain the experimental data. We have therefore chosen to summarize quantitative parameters using the grading system --, +, ++ and +++, as indicated in the footnotes. In some cases these grades had to be deduced from the primary reports, so readers are encouraged to consult the original papers for quantitative details; we regret any errors of interpretation we may have made during this estimation process. Not surprisingly, perhaps, different studies sometimes yielded conflicting results. We have recorded the conflicting data sets but shall leave it to the readers to judge which are the more plausible.

The natural variability of residues in clade B and C isolates was analyzed and mapped on the structure of gp41 (see Figure 2 and Figure 3). A focus of variable residues in clade B sequences is located in the upper part of the C-terminal helix centered around the highly variable leucine-glutamate-glutamine (LEQ) triplet, indicating that this region is under selective pressure. However, it is also possible that certain changes in residues in this region have little impact on Env function, particularly if there is some flexibility in Env structure(s) around this region. This relatively variable region also contains four glycosylation sites, which could be involved in immune evasion (30). Indeed, mutations that affect glycosylation in this region can modulate neutralization sensitivity (65). Of note is that no CTL or antibody epitopes have been mapped to this region despite the intense positive selection. One interpretation of this observation is that the selection pressure is exerted indirectly on distant antibody epitopes elsewhere in gp41e or even in gp120 (32). Another is that some neutralizing antibodies remain as yet undiscovered in this region of gp41e. In clade C viruses the variability is somewhat shifted towards the 2F5 epitope, compared to clade B. Furthermore, certain residues are significantly more variable in clade C viruses compared to clade B, and vice versa, suggesting that subtly different selection pressures may operate on viruses from the two clades.

Acknowledgments

We thank Brian Foley and Charles Calef for their help with graphical presentation of Figures and Tables. Financial support was obtained from the Dutch AIDS Fund, Amsterdam.

References

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Figures and Table

 

            gp41 start, position 512 of HXB2 gp160
            |
            AVGIGALFL GFLGAAGSTM GAASMTLTVQ ARQLLSGIVQ 550
QQNNLLRAIE AQQHLLQLTV WGIKQLQARI LAVERYLKDQ QLLGIWGCSG 600
KLICTTAVPW NASWSNKSLE QIWNHTTWME WDREINNYTS LIHSLIEESQ 650
NQQEKNEQEL LELDKWASLW NWFNITNWLW YIKLFIMIVG GLVGLRIVFA 700
VLSIVNRVRQ GYSPLSFQTH LPTPRGPDRP EGIEEEGGER DRDRSIRLVN 750
GSLALIWDDL RSLCLFSYHR LRDLLLIVTR IVELLGRRGW EALKYWWNLL 800
QYWSQELKNS AVSLLNATAI AVAEGTDRVI EVVQGACRAI RHIPRRIRQG 850
LERILL 856

Figure 1. The HXB2 reference strain and the numbering of positions in the gp41 sequence. Only information on the ectodomain (residues 512-684) is incorporated in subsequent analyses.

 

Figure 2. Variability of gp41e. The relative entropies of residues were mapped onto a 2D representation of the HXB2 gp41e (adapted from 29, 61). The variability of residues in clade B isolates (left panel) and clade C isolates (right panel) is indicated according to their entropy values. The entropy is a simple measure of variation in each position based on a sequence alignment (33). Not surprisingly, entropy values for each amino acid were highly correlated with the ratio of the nonsynonymous/synonymous substitution rates, a measure which is indicative of selective pressure, calculated using PAML (76) (Spearman's rank correlation tests gave z = 7.3, p = 2 x 10-13 for the B clade, and z = 7.5, p = 5 x 10-14 for the C clade). We used the entropy scores as our measure of variability here because they lent themselves to testing for differences in variability between the B clade and C clade (33). The color coding for the sites is as follows: white, invariant (entropy score of zero); light blue, very conserved (entropy score below the median, corresponding to only one observed substitution); medium blue, variable (entropy score above the median: 2 or more observed substitutions); dark blue, highly variable (highest 10% of entropy scores: > 0.8 for clade B and > 0.75 for clade C). Residues that are significantly more variable in clade B than in clade C or vice versa (p value <= 0.03 after a Bonferroni correction for multiple tests, using a Monte Carlo scheme and randomizing the B and C clade data 10,000 times) are indicated by red circles. 123 clade B sequences and 48 clade C sequences were used for the analyses. The four glycans and the major antibody epitopes (non-neutralizing clusters I and II and the neutralizing 2F5/4E10/z13 cluster) are also indicated, as are regions labeled "indel" where insertions and deletions are frequently observed in natural variants.

 

Figure 3. The residues with the highest 10% of entropy scores in clade B are indicated in red on the 3D structure model of Caffrey (pdb accession number 1IF3, (8)). These residues are only indicated in one monomer. The other two monomers are shown in grey for orientation purposes.

Table 1

Residue1 Comments Substitution Isolate2 Reference Expression (cell lysate)3 Expression (cell surface)4 gp160 processing5 gp120 association6 CD4-binding7 CD4-induced shedding8 Cell-cell fusion9 Virion incorporation10 Virus entry11 Viral Replication12 Oligomerization (gp160/gp140)13 Trimerization (SOS gp140)14 Thermal stability (gp41 core)15 B-clade entropy C-clade entropy
WT ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++
A512 V16 NL4-3 Freed 1990 ++ ++ ++ ++ + 0.136 0
E NL4-3 Freed 1990 ++ ++ ++ ++ +
V513 E NL4-3 Freed 1990 ++ ++ ++ ++ - 0.326 0.44
A NL4-3 Buchschacher 1995 ++
G NL4-3 ++
R NL4-3 ++ ++ -
G514 V NL4-3 Delahunty 1996 ++ ++ ++ ++ 0.628 0.594
G516 V NL4-3 Delahunty 1996 +++ ++ ++ ++ 0.047 0.101
A517 17 HXB2 Kowalski 1991 ++ ++ ++ ++ ++ ++ 0.115 0
18 HXB2 Kowalski 1991 -
M518 V19 ELI1 Kozak 1997 +++ ++ 0.985 0.658
F519 L16 NL4-3 Freed 1990 ++ ++ ++ ++ + 0.19 0.473
V NL4-3 Delahunty 1996 +++ ++ ++ ++ ++
L520 R NL4-3 Freed 1990 ++ ++ ++ ++ - 0.13 0.101
G521 V NL4-3 Delahunty 1996 + ++ ++ ++ - - 0 0
F522 V NL4-3 Delahunty 1996 +++ ++ ++ + 0 0.302
G BH8 Pritsker 1999 ++ ++ +
G524 V NL4-3 Delahunty 1996 +++ ++ ++ ++ + + 0.083 0.101
A525 T20 LAI Bahbouhi 2001 ++ ++ ++ ++ 0.115 0.202
A526 E NL4-3 Freed 1990 ++ + + ++ - 0 0
G527 V NL4-3 Delahunty 1996 +++ ++ - - 0 0
S528 T HXB2 Cao 1993 + + - + - + 0 0
M530 S HXB2 Cao 1993 ++ - - + - - 0 0
G531 V NL4-3 Delahunty 1996 +++ ++ ++ ++ 0 0
L537 R NL4-3 Freed 1990 ++ + + ++ - 0 0
V539 E NL4-3 Freed 1990 ++ ++ ++ ++ + 0.083 0.334
Q540 L NL4-3 Freed 1990 ++ + + ++ - 0 0
R542 e in heptad-repeat G NL4-3 Freed 1990 ++ ++ ++ ++ + 0 0.101
Q543 f in heptad-repeat H PI Wei 2002, Kilby 2002 ++ 0.811 0.202
R ++
P543 L28 MN Park 2000 ++
L544 g in heptad-repeat S22 PI Fikkert 2002 ++ 0.094 0.234
L545 a in heptad-repeat F21 JR-FL Sanders 2002 - + 0.047 0
N21 JR-FL ++ +
P21 JR-FL ++ +
G21 JR-FL ++ +
G547 c in heptad-repeat S22 NL4-3 Rimsky 1998 ++
D22 NL4-3 ++ 0 0.101
D22 PI Baldwin 2003 ++
V22 PI Poveda 2002 ++
D22 PI Wei 2002 ++
I548 d in heptad-repeat A HXB2 Cao 1993 ++ ++ + +++ ++ + ++ 0 0.101
T22 NL4-3 Rimsky 1998 ++
K22 PI Baldwin 2003 ++
V22 PI Wei 2002, Kilby 2002 ++
V21 JR-FL Sanders 2002 ++ +
L21 JR-FL ++ +
H21 JR-FL ++ +
N21 JR-FL ++ +
S21 JR-FL ++ +
G21 JR-FL ++ +
R21 JR-FL ++ +
V549 e in heptad-repeat M22 NL4-3 Rimsky 1998 ++ 0.047 0
M22 PI Wei 2002 ++
A22 PI ++
A22 PI Baldwin 2003 ++
W22 PI ++
G22 PI ++
A HXB2 Lu 2001 ++ ++ ++ ++ ++
Q551 g in heptad-repeat A HXB2 Lu 2001, Follis 2002 ++ ++ ++ ++ ++ ++ 0 0
Q552 a in heptad-repeat L HXB2 Cao 1993 ++ - - - 0 0
N554 c in heptad-repeat K22 PI Fikkert 2002 ++ 0.047 0
L555 d in heptad-repeat G HXB2 Cao 1993 ++ - - - 0 0
A BH8 Poumbourios 1997 ++ - ++ - ++
V21 JR-FL Sanders 2002 -
W21 JR-FL -
Y21 JR-FL -
S21 JR-FL -
P21 JR-FL -
L556 e in heptad-repeat P HXB2 Chen 1994 ++ + - - 0.047 0
R HXB2 Weng 1998 - - -
E HXB2 - - -
A HXB2 + - -
D HXB2 Weng 2000 ++ ++ ++ -
G HXB2 ++ ++ ++ -
K HXB2 - -
N HXB2 ++ ++ ++ -
A HXB2 Lu 2001, Follis 2002 ++ + ++ - + ++
P21 JR-FL Sanders 2002 ++ +
R557 f in heptad-repeat P21 JR-FL Sanders 2002 ++ + 0.237 0.334
M PI Wei 2002 ++
A558 g in heptad-repeat R HXB2 Weng 1998 - - 0 0
E HXB2 + -
C HXB2 Weng 2000 ++ ++ ++ -
G HXB2 ++ ++ ++ -
T HXB2 ++ ++ ++ +
P21 JR-FL Sanders 2002 ++ +
I559 a in heptad-repeat P HXB2 Chen 1993, Chen 1994 ++ ++ - - ++ - - ++ 0.047 0
A BH8 Poumbourios 1997 ++ - ++ - ++
V21 JR-FL Sanders 2002 + +
F21 JR-FL - +++
N21 JR-FL - +++
P21 JR-FL ++ ++ +++ -
G21 JR-FL + ++ +++ +
R21 JR-FL + +++
P LAI/JR-FL Sanders 2003b - -
G LAI/JR-FL - +
L LAI/JR-FL ++ ++
E560 b in heptad-repeat P21 JR-FL Sanders 2002 +++ + 0.217 0
G19 ELI1 Kozak 1997 +
A561 c in heptad-repeat P21 JR-FL Sanders 2002 +++ + 0.094 0.101
S561 A28 MN Park 2000 ++
Q562 d in heptad-repeat L HXB2 Cao 1993 ++ + - - 0 0.101
A BH8 Poumbourios 1997 ++ ++ + ++ - ++
P21 JR-FL Sanders 2002 +++ +
Q563 e in heptad-repeat A HXB2 Weng 2000 ++ ++ ++ ++ 0.047 0
E HXB2 ++ ++ ++ -
M HXB2 ++ ++ ++ -
G HXB2 ++ ++ ++ ++
R HXB2 ++ ++ ++ +
A HXB2 Lu 2001, Follis 2002 ++ ++ ++ ++ ++ ++
P21 JR-FL Sanders 2002 +++ +
R564 f in heptad-repeat P21 JR-FL Sanders 2002 +++ + 0.047 0
H564 N28 MN Park 2000 ++
C26 HXB2 Rabenstein 1995 ++
L565 g in heptad-repeat P HXB2 Chen 1994 ++ ++ + ++ ++ - 0.402 0.584
A HXB2 Lu 2001, Follis 2002 ++ ++ ++ - - +
P21 JR-FL Sanders 2002 ++ +
L566 a in heptad-repeat G HXB2 Cao 1993 ++ + + - - + 0.047 0
P HXB2 Chen 1993, Chen 1994 ++ ++ + + ++ - - ++
A BH8 Poumbourios 1997 ++ - ++ - ++
V23 BH8 Earl 1993 ++ ++ ++ ++
V21 JR-FL Sanders 2002 + ++ ++ ++
I21 JR-FL - +
N21 JR-FL + ++
T21 JR-FL + ++
P21 JR-FL + ++ +
K21 JR-FL - +
Q567 b in heptad-repeat R LAI Sanders 2003a ++ ++ 0.177 0
L568 c in heptad-repeat A HXB2 Cao 1993 ++ ++ + ++ ++ ++ - + 0 0
P HXB2 Chen 1994 ++ ++ + + ++ -
A HXB2 Ji 2000 +
T569 d in heptad-repeat A BH8 Poumbourios 1997 ++ - ++ - ++ 0 0.101
C HXB2 Farzan 1998 -
S21 JR-FL Sanders 2002 + +
P21 JR-FL + ++ ++ +
K21 JR-FL + ++
E21 JR-FL -
V570 e in heptad-repeat R HXB2 Weng 1998 ++ ++ ++ - ++ - 0 0
E HXB2 ++ ++ ++ ++ ++ ++
A HXB2 Weng 2000 ++ ++ ++ -
D HXB2 ++ ++ ++ -
E HXB2 ++ ++ ++ -
G HXB2 ++ ++ ++ -
I HXB2 ++ ++ ++ ++
A HXB2 Lu 2001, Follis 2002 ++ ++ ++ - - +
W571 f inheptad-repeat R HXB2 Cao 1993 ++ ++ + ++ ++ - - - 0 0
R HXB2 Ji 2000 ++
C26 HXB2 Rabenstein 1995 ++
G572 g in heptad-repeat G HXB2 Weng 1998 ++ - ++ - - - 0 0
A HXB2 Lu 2001 ++ ++ ++ - +++
I573 a in heptad-repeat L HXB2 Dubay 1992 ++ ++ ++ ++ ++ ++ ++ 0.083 0
V HXB2 ++ ++ ++ ++ ++ ++ ++ ++
A HXB2 ++ ++ ++ + ++ + ++
G HXB2 ++ ++ ++ ++ - ++ - ++
E HXB2 ++ ++ ++ ++ - ++ - ++
D HXB2 ++ ++ ++ - ++ - ++
S HXB2 ++ ++ ++ - ++ - ++
P24 HXB2 Bernstein 1995 -
A24 HXB2 +
D24 HXB2 -
A25 HXB3 Shugars 1996 ++
S25 HXB3 -
P HXB2 Chen 1993, Chen 1994 ++ +++ + + ++ - - ++
P26 HXB2, LAI Wild 1994 ++ ++ + - -
A26 HXB2, LAI ++ ++ ++ ++ + + -
S26 HXB2, LAI ++ ++ ++ ++ - - -
P26 HXB2 Rabenstein 1995 -
D26 HXB2 -
S26 HXB2 -
S 168P Liu 2001 -
T 168P ++ ++ ++ ++ ++ ++ +
V LAI Sanders 2003a ++ ++
A BH8 Poumbourios 1997 ++ ++ ++ ++ - ++
V HXB2 Markosyan 2002 ++
A HXB2 +
S HXB2 +
P HXB2 -
L21 JR-FL Sanders 2002 ++ +
F21 JR-FL ++ +
Y21 JR-FL ++ +
Q21 JR-FL ++ +
N21 JR-FL ++ +
T21 JR-FL ++ +
P21 JR-FL ++ +
G21 JR-FL ++ +
K21 JR-FL ++ +
K574 b in heptad-repeat R BH8 McInerney 1998 ++ ++ ++ ++ ++ + 0 0
L576 d in heptad-repeat P HXB2 Chen 1994 ++ + + ++ - 0 0
A BH8 Poumbourios 1997 ++ - ++ - ++
C27 HXB2 Farzan 1998 ++ + - +++
V21 JR-FL Sanders 2002 - +
F21 JR-FL - +
Y21 JR-FL - +
Q21 JR-FL - +
N21 JR-FL - +
G21 JR-FL - +
K21 JR-FL - +
Q577 e in heptad-repeat R HXB2 Weng 1998 ++ ++ ++ ++ - 0.047 0.173
E HXB2 ++ ++ ++ + + +
A HXB2 Weng 2000 ++ ++ ++ +
D HXB2 ++ ++ ++ ++
E HXB2 ++ ++ ++ +
G HXB2 ++ ++ ++ ++
M HXB2 ++ ++ ++ +
C27 HXB2 Farzan 1998 ++ + - +++
A HXB2 Lu 2001 ++ ++ ++ ++ ++
A578 f in heptad-repeat G27 HXB2 Farzan 1998 ++ + - +++ 0.047 0.483
R579 g in heptad-repeat G HXB2 Weng 2000 ++ + ++ - 0 0.101
A HXB2 Lu 2001 ++ + ++ - ++
I580 a in heptad-repeat P HXB2 Chen 1994 ++ ++ + - ++ - 0.432 0.173
A BH8 Poumbourios 1997 ++ ++ ++ ++ - ++
L21 JR-FL Sanders 2002 ++ +
H21 JR-FL ++ +
T21 JR-FL ++ +
P21 JR-FL ++ +
G2 JR-FL ++ +
L581 b in heptad-repeat Q28 MN Park 2000 ++ 0 0
A582 c in heptad-repeat T28 PI Reitz 1988 ++ 0 0
C26 HXB2 Rabenstein 1995 ++
V583 d in heptad-repeat A BH8 Poumbourios 1997 ++ + ++ ++ - ++ 0.244 0.503
C HXB2 Farzan 1998 -
L21 JR-FL Sanders 2002 ++ +
Q21 JR-FL ++ +
N21 JR-FL ++ +
S21 JR-FL ++ +
P21 JR-FL ++ +
R21 JR-FL ++ +
K21 JR-FL ++ +
E584 e in heptad-repeat A HXB2 Cao 1993 ++ - - + - - 0 0
Q BH8 Maerz 2001 ++ ++ ++ + +
D BH8 ++ ++ +
N BH8 ++ ++ -
Y586 f in heptad repeat R HXB2 Weng 1998 ++ + ++ + - 0 0.101
E HXB2 ++ + ++ + -
C29 HXB2 Farzan 1998 -
L587 a in heptad-repeat P HXB2 Chen 1993, Chen 1994 ++ ++ ++ - ++ - - ++ 0 0
A BH8 Poumbourios 1997 ++ ++ ++ ++ - ++
C29 HXB2 Farzan 1998 -
A21 JR-FL Sanders 2002 - +
P21 JR-FL - +
R21 JR-FL - +
D21 JR-FL - +
E21 JR-FL - +
K588 R BH8 McInerney 1998 ++ ++ ++ ++ ++ ++ 1.112 0.775
D589 L HXB2 Cao 1993 ++ ++ +++ + ++ + - + 0 0.101
C30 JR-FL Binley 2000 ++
K BH8 Maerz 2001 ++ ++ ++ + -
Q591 A BH8 Maerz 2001 ++ ++ ++ ++ ++ 0.083 0.101
K BH8 ++ ++ ++
L LAI Sanders 2003c +
L592 V BH8 Maerz 2001 ++ ++ ++ 0 0.101
A BH8 ++ ++ ++
L593 V BH8 Maerz 2001 ++ ++ + - 0.143 0
A BH8 ++ ++ ++ - + -
Q LAI Sanders 2003c +/-
I595 F31 PI Moore 1993 ++ 0.162 0.555
W596 M HXB2 Cao 1993, Cao 1994 ++ ++ ++ + ++ - ++ ++ ++ 0 0
Y LAI, NL4-3 Rovinski 1999 ++ ++
A LAI, NL4-3 - +
C30 JR-FL Binley 2000 ++
F BH8 Maerz 2001 ++ ++ ++ + ++ ++
H BH8 ++ ++ +
L BH8 ++ ++ ++ + +
G597 P BH8 Maerz 2001 ++ ++ ++ - - 0 0
A BH8 ++ ++ ++ - -
S BH8 ++ ++ ++ - -
C598 S HXB2 Dedera 1992a ++ - - 0 0
S23 BH8 Earl 1993 ++ ++ ++ ++
G HXB2 Syu 1991 ++ - -
A LAI Van Anken 2003 -
G600 A LAI, NL4-3 Rovinski 1999 ++ ++ 0 0.101
K601 R BH8 McInerney 1998 ++ ++ ++ ++ ++ ++ 0.218 0
R LAI, NL4-3 Rovinski 1999 ++ ++
E LAI, NL4-3 ++ ++
E BH8 Merat 1999 ++ + ++
E BH8 Maerz 2001 ++ ++ ++ + ++
H BH8 ++ ++ + ++
Q BH8 ++ ++ + ++
A BH8 ++ ++ ++
C604 S HXB2 Dedera 1992a ++ - - 0.047 0
S23 BH8 Earl 1993 ++ ++ ++ ++
G HXB2 Syu 1991 ++ - -
A LAI Van Anken 2003 -
T605 C30 JR-FL, HXB2, DH123, 89.6, GUN1-wt Binley 2000 ++ ++ +++ ++ + 0.177 0.173
C LAI Sanders 2003c ++
Y LAI ++
V608 S HXB2 Cao 1993 - - - 0.094 0.101
C30 JR-FL Binley 2000 ++
P609 C30 JR-FL Binley 2000 ++ 0.047 0.101
W610 C30 JR-FL Binley 2000 ++ 0.047 0
F BH8 Maerz 2001 ++ ++ ++ - -
H BH8 ++ ++ ++ - -
N611 Glycosylation site Q HXB2 Dedera 1992b ++ ++ ++ + ++ ++ 0.141 0
H HXB2 Lee 1992 ++ ++ ++ +
S NL4-3 Dash 1994 ++ ++ ++ ++
Q SHIV-KB9 Johnson 2001 ++ ++ ++
S613 Glycosylation site N611 A HXB2 Lee 1992 ++ ++ + 0.94 0.274
N616 Glycosylation site Q HXB2 Dedera 1992b + ++ 0.237 0
Q23 BH8 Earl 1993 ++ ++ ++ ++
H HXB2 Lee 1992 ++ ++ ++ ++
S NL4-3 Dash 1994 ++ ++ ++ ++
Q BH10 Perrin 1998 ++ ++ +
Q SHIV-KB9 Johnson 2001 ++ ++ ++
K617 R BH8 McInerney 1998 ++ ++ ++ ++ ++ ++ 0.348 0.658
S618 Glycosylation site N616 A HXB2 Lee 1992 ++ ++ - - 0.495 0.483
N624 d in heptad-repeat Glycosylation site (N625 in most isolates) H HXB2 Lee 1992 ++ ++ ++ + 1.153 1.305
Q BH10 Perrin 1998 ++ ++ ++
Q SHIV-KB9 Johnson 2001 ++ ++ ++
N625 e in heptad-repeat Glycosylation site Q23 BH8 Earl 1993 ++ ++ ++ ++ 0.047 0.274
T626 f in heptad-repeat Glycosylation site N624 M HXB2 Cao 1993 ++ - - - - - - 0.244 0.444
M28 SHIV-HXBc2P Si 2001 ++
W628 a in heptad-repeat M HXB2 Cao 1993 - - - - 0 0
A HXB2 Weng 2000 ++ - ++ -
F HXB2 ++ - ++ -
A HXB2 Wang 2002 ++ - ++ - +
W631 d in heptad-repeat A HXB2 Wang 2002 ++ - ++ - - 0 0.101
D632 e in heptad-repeat N32 BH10 Perrin 1998 ++ ++ - 0.591 0.287
R633 f in heptad-repeat G PI Wei 2002 ++ 0.55 0.451
I635 a in heptad-repeat A HXB2 Wang 2002 ++ - ++ - + 0.047 0.173
N637 c in heptad-repeat Glycosylation site K22 PI Baldwin 2003 ++ 0.141 0.101
Q HXB2 Dedera 1992b ++ ++
Q23 BH8 Earl 1993 ++ ++ ++ ++
H HXB2 Lee 1992 ++ ++ ++ -
S NL4-3 Dash 1994 ++ - - -
Q BH10 Perrin 1998 ++ ++ ++
Q SHIV-KB9 Johnson 2001 ++ ++ ++
Y638 d in heptad-repeat A HXB2 Wang 2002 ++ ++ ++ ++ ++ 0.13 0
T639 e in heptad-repeat Glycosylation site N637 V HXB2 Lee 1992 ++ ++ + 0.083 0.202
A HXB2 Cao 1993 ++ - - - -
I642 a in heptad-repeat A HXB2 Wang 2002 ++ - ++ - ++ 0.094 0
A HXB2 Markosyan 2002 ++
S HXB2 ++
H643 b in heptad-repeat Y20 LAI Bahbouhi 2001 ++ ++ ++ ++ 0.115 0
Y LAI Sanders 2003a ++
L645 d in heptad-repeat A H64333 Wang 2002 ++ ++ ++ ++ ++ 0 0
E647 f in heptad-repeat L HXB2 Cao 1993 ++ +++ + ++ ++ 0.188 0.173
S649 a in heptad-repeat A HXB2 Wang 2002 ++ ++ ++ ++ ++ 0.401 0
Q652 d in heptad-repeat L HXB2 Cao 1993 ++ ++ + ++ ++ 0.047 0.101
L HXB2 Shu 2000 ++
A HXB2 Wang 2002 ++ ++ ++ ++ ++
K655 g in heptad-repeat R33 BH8 Poumbourios 1995 ++ ++ ++ ++ 0.213 1.093
N656 a in heptad-repeat L HXB2 Cao 1993 ++ ++ + ++ ++ ++ - + 0 0
L663 2F5 epitope F HXB2 Cao 1993 ++ +++ ++ ++ ++ 0.047 0.101
K665 2F5 epitope R33 BH8 Poumbourios 1995 ++ ++ ++ ++ 0.451 0.922
W666 2F5 epitope P HXB2 Cao 1993 ++ ++ ++ ++ ++ 0.047 0.101
A HXB2, NL4-3 Salzwedel 1999 ++ ++ ++ ++
S668 2F5 epitope N28 HXB2 Back 1993 ++ 0.497 0.573
L669 P HXB2 Cao 1993 +++ ++ + +++ ++ ++ 0.047 0
W670 A HXB2, NL4-3 Salzwedel 1999 ++ ++ ++ ++ 0.047 0
N671 4E10/z13 epitope P HXB2 Cao 1993 ++ ++ ++ ++ ++ 0.713 0.945
W672 4E10/z13 epitope S HXB2 Cao 1993 ++ ++ ++ + +++ ++ ++ 0 0
S HXB2, NL4-3 Salzwedel 1999 ++ ++ ++ ++
P HXB2, NL4-3 ++ ++ ++ ++ - +
F HXB2, NL4-3 ++ ++ ++ ++ + +
F673 4E10/z13 epitope P HXB2 Cao 1993 ++ ++ ++ + ++ ++ 0.94 0
S34 HXB2 Stern 1995 ++ ++
N674 4E10/z13 epitope H HXB2 Lee 1992 ++ ++ ++ ++ 1.038 1.375
S NL4-3 Dash 1994 ++ ++ ++ ++
D28 SHIV-HXBc2P Si 2001 ++
I675 4E10/z13 epitope S HXB2 Cao 1993 ++ ++ + ++ ++ 0 0
M28 HXB2 Back 1993 ++
N677 R HXB2 Cao 1993 ++ ++ + ++ ++ 1.237 0.769
W678 A HXB2 Cao 1993 ++ ++ + ++ ++ 0 0
A HXB2, NL4-3 Salzwedel 1999 ++ ++ ++ ++
W680 A HXB2, NL4-3 Salzwedel 1999 ++ ++ ++ ++ 0.047 0.101
Y681 P HXB2 Cao 1993 ++ ++ ++ ++ 0 0
K683 R BH8 McInerney 1998 ++ ++ ++ ++ ++ ++ 0.375 0.325

Footnotes

1Residue numbering is based on HXB2 gp160, although the amino-acids studied may be different in the isolate used. The one-letter code for amino acids is used
2PI: primary isolate
3As assessed by western blot or immunoprecipitation. -, minimal or no expression; +, reduced expression; ++, expression similar to WT; +++, increased expression
4As assessed by surface biotinylation, iodination or FACS. When soluble gp140 constructs were used, the relative secretion levels (western blot or immunoprecipitation) are given. -, minimal or no expression; +, reduced expression; ++, expression similar to WT; +++, increased expression
5As assessed by western blot or immunoprecipitation in combination with densitometric measurements. -, minimal or no processing; +, reduced processing; ++, processing similar to WT; +++, increased processing
6As assessed by western blot or immunoprecipitation in combination with densitometric measurements. -, minimal or no association; +, reduced association; ++, association similar to WT; +++, increased association
7As assessed by immunoprecipitation with CD4-based reagents. ++, similar to WT; +++, increased CD4 binding
8As assessed by immunoprecipitation. -, no shedding; +, reduced shedding; ++, shedding similar to WT; +++, increased shedding. Note that CD4-induced shedding and to a lesser extent gp120 association (i.e., the reverse of shedding), when measured in laboratory isolates, might be diminished in primary isolates that can retain gp120 more efficiently.
9As assessed by syncytium formation or reporter gene assays. -, fusion lower than 3% of WT; +, fusion between 3 and 30% of WT; ++, fusion greater than 30% of WT
10As assessed by western blot or immunoprecipitation. -, minimal or no incorporation; +, reduced incorporation; ++, incorporation similar to WT
11As assessed by various assays (replication complementation, use of reporter genes, p24 production). -, entry lower than 3% of WT; +, entry between 3 and 30% of WT; ++, entry greater that 30% of WT
12-, no apparent replication; +, replication with a delay of more than 2 days compared to WT; ++ replication similar to WT
13As assessed by sucrose gradient fractionation, immunoprecipitation, velocity sedimentation or FPLC, unless indicated otherwise. -, oligomerization below 25% of WT; +, oligomerization between 25% and 50% of WT; ++, oligomerization similar to WT. No distinction between dimerization, trimerization or tetramerization is made.
14As assessed by Blue Native-PAGE. +, trimerization similar to WT SOS gp140 (occasional trimerization); ++, slightly more trimerization than in WT; +++, significantly more trimerization than in WT.
15As analyzed using the N34(L6)C28 or N36(L6)C34 peptide model, unless indicated otherwise. -, melting temperature (Tm) below 40 degrees C; +, Tm between 40 degrees C\ and 60 degrees C; ++, Tm between 60 degrees C\ and 80 degrees C; +++, Tm over 80 degrees C
16Analyzed in a double mutant, A512V + F519L
17Four amino-acid insertion GIPA
18Six amino-acid insertion IHRWIA
19Involved in cell line adaptation
20Identified in an isolate which is resistant to the furin inhibitor (alpha1-PDX)
21Analyzed in soluble SOS gp140 constructs and so also contain the A501C and T605C substitutions
22Involved in T-20 resistance
23Analyzed in soluble gp140
24Analyzed in an N-peptide/Protein A fusion protein
25Analyzed in an N-peptide/maltose binding protein (MBP) fusion protein
26Thermal stability (74) or oligomerization (53) of N-peptides analyzed in the absence of C-peptides
27Analyzed in a triple mutant L576C + Q577C + A578G
28Involved in neutralization resistance
29Analyzed in a double mutant Y586C + L587C
30Analyzed in combination with gp120 cysteine substitutions in the context of soluble gp140
31Involved in resistance to soluble CD4
32Generates a new glycosylation site
33Analyzed in a double mutant K655R + K665R
34Analyzed in a double mutant A582T + F673S
35Data on this mutant were corrected in reference 73
last modified: Wed Jan 13 14:58 2010


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