Human Immunodeficiency Virus Type 1 gpl20 Envelope Glycoprotein Regions Important for Association with

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1 JOURNAL OF VIROLOGY, Apr. 1991, p X/91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 65, No. 4 Human Immunodeficiency Virus Type 1 gpl20 Envelope Glycoprotein Regions Important for Association with the gp4l Transmembrane Glycoprotein EIRIK HELSETH, UDY OLSHEVSKY, CRAIG FURMAN, AND JOSEPH SODROSKI* Division of Human Retrovirology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, 44 Binney Street, Boston, Massachusetts Received 18 October 1990/Accepted 7 January 1991 Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gpl20 or gp4l envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237: , 1987). To localize the determinants on the gpl20 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gpl20 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gpl20 regions. These results suggest that hydrophobic regions at both ends of the gpl20 glycoprotein contribute to noncovalent association with the gp4l transmembrane glycoprotein. Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS (1, 4, 18), which is characterized by depletion of CD4+ lymphocytes (5, 12). The tropism of HIV-1 for CD4+ cells is due to a specific interaction between CD4, the viral receptor, and the gp120 exterior envelope glycoprotein (3, 7, 8, 15). Following receptor binding, the viral envelope glycoproteins gpl20 and gp4l mediate the fusion of the viral and host cell membranes to allow virus entry (21). The HIV-1 envelope glycoproteins expressed in infected cells also mediate some of the cytopathic effects accompanying viral infection. Interaction of HIV-1 envelope glycoproteins with the CD4 molecule has been implicated in syncytium formation and single-cell lysis (9, 14, 20). The HIV-1 gpl20 exterior envelope glycoprotein and the gp4l transmembrane envelope glycoprotein are derived from a gp160 precursor glycoprotein. Following proteolytic cleavage of the gpl60 envelope glycoprotein precursor, the gpl20 exterior glycoprotein associates with the gp4l transmembrane glycoprotein via noncovalent interactions (10). Up to 50% of newly synthesized gp120 can be detected in the supernatant of cells expressing the wild-type HIV-1 envelope glycoproteins, suggesting that the association between gpl20 and gp4l is labile. Insertion of four amino acids into various locations within the amino-terminal half of the gp120 or gp4l glycoprotein resulted in nearly complete dissociation of the gp120 and gp4l subunits (10). The gpl20 moieties of the mutant envelope glycoproteins were found primarily in the supernatant of envelope-expressing cells, whereas the levels of cellassociated gpl20 were significantly reduced compared with those of the wild-type protein. While the mutant gpl20 molecules bound CD4, the ability of the envelope glycoproteins to induce the formation of syncytia or to allow virus replication was significantly attenuated (6, 10). * Corresponding author To define HIV-1 gpl20 glycoprotein regions important for association with the gp4l transmembrane glycoprotein, a number of gpl20 residues were changed by site-directed mutagenesis of the HIV-1 env gene as described before (6, 11, 17). These residues were selected because they either did not vary or exhibited strong conservation of features among HIV-1, HIV-2, and simian immunodeficiency viruses (SIVs) of macaques and African green monkeys (SIVmac and SIVagm, respectively) (16). Most of the changes, listed in Table 1, consisted of nonconservative substitutions. At least two independently derived clones of each mutated env gene were evaluated for phenotype. The clones of each mutated env gene were introduced into the psviiienv plasmid, which allows transient expression of the env gene in transfected COS-1 cells (6). Transfected cells were continuously radiolabeled with [35S]cysteine for 12 to 14 h, and steady-state levels of envelope glycoprotein expression were assessed by precipitation of cell lysates and supernatants with an excess of serum from an AIDS patient (19501) as described previously (2, 6). To compare the ability of different gpl20 mutants to associate with the gp4l transmembrane glycoprotein, the levels of envelope glycoproteins in the lysates and supernatants of the transfected cells were measured by densitometry and used to calculate the association index. The association index is a measure of the ability of the mutant gpl20 molecule to remain associated with the expressing cell relative to that of the wild-type glycoprotein and is calculated as follows: association index = ([mutant gpl20]cel X [wild-type gp120]supematant)/([mutant gpl2lsupernatant x [wild-type gpl20ic,e11). Since some mutations affecting the exterior domain of the gp4l glycoprotein have been shown to result in complete loss of gpl20 cell association (10), the ability of the gpl20 molecule to associate with the expressing cell is dependent upon interaction with gp4l. Furthermore, differences in the rate of cell surface transport among mutant envelope glycoproteins would not contribute significantly to differences in

2 2120 NOTES J. VIROL. TABLE 1. Association indices of HIV-1 gp120 mutants Amino acid Association Amino acid Association Amino acid Association changea indexb changea index' changea indexb None (wild type) N/T V/S V/L A/E K/A Y/D E/L A/L W/S E/L Y/H W/L R/G Y/S P/Y /309/310 RIQ/RPELIPVQ P/R N/R G/W S/I N/P D/T R/K E/R D/R D/A E/L E/Q D/R Q/F E/R N/D E/A N/K P/L D/A G/F P/G D/R /381 GE/YV M/S K/W E/P D/V /121 VK/LE F/L D/S L/G Y/E /483/484 ELY/GRA...< K/W W/S K/V R/W I/R I/F S/Y K/L P/K T/R W/V G/K T/A W/S /498/499 APT/VLL...< T/G K/L /501 KA/KGIPKA The SalI-BamHI fragment of the psviiienv plasmid was used for site-directed mutagenesis by the procedure of Kunkel et al. (11). The presence of the mutation was confirmed by the generation of a novel restriction endonuclease site in some cases and by DNA sequencing. The number of the mutant refers to the envelope glycoprotein amino acid residue of the HXBc2 strain of HIV-1, where 1 is the initial methionine (19). The mutations result in substitution of the amino acid(s) shown on the right for the amino acid(s) shown on the left of the slash; for example, 273 R/I indicates a substitution of isoleucine for the arginine residue at position 273. The mutations affecting residues 308/309/310 and 500/501 result in the insertion of additional amino acids into the primary sequence. b Immunoprecipitates of radiolabeled cell lysates and supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiograms were analyzed by densitometry, and the association index was calculated as described in the text. For the mutant phenotypes reported, at least two independently mutated plasmids yielded results in transfected COS-1 cells that did not vary by more than 20% of the value reported. x a) -o c 0 * (I) C,) Iu 1 0) 0 -j SEC [~ 0 V1/ C3 V3 V4 C4 500 C5 V5

3 VOL. 65, 1991 B >i 3: _ C s C S C S C) ~ A > C S C S C S E.L. b~i 6 P a Lr =2) cc 2_ -Z v X C S C S C S C S cl, cn C S C S cn, sr C) rv 1 3 I -160 FIG. 2. Steady-state levels of envelope glycoprotein mutants in transfected cells. (A) Immunoprecipitates of envelope glycoproteins from cell lysates (lanes 1 to 3) and supernatants (SUP, lanes 4 to 6) of COS-1 cells mock transfected with no DNA (lanes 1 and 4) or transfected with a plasmid expressing the wild-type (w.t.) envelope glycoproteins (lanes 2 and 5) or the 500/501 KA/KGIPKA mutant (lanes 3 and 6). The positions of the gp160, gpl20, and gp41 glycoproteins are shown. (B) Immunoprecipitates of envelope glycoproteins from cell lysates (C) or supernatants (S) of COS-1 cells that were mock transfected (Mock, lanes 1) or transfected with a plasmid expressing the wild-type (w.t.) envelope glycoproteins (lanes 2) or the indicated mutant proteins. Note that a protein band unrelated to the HIV-1 envelope glycoproteins is seen in the supernatants of all, even mock-transfected, COS-1 cells in this experiment. The positions of gpl60 and gp120 are indicated. association indices observed for this long labeling period. Thus, the association index provides an indirect measure of the interaction of mutant gpl20 glycoproteins with the gp4l transmembrane glycoprotein. The association indices calculated for the gpl20 mutants are listed in Table 1 and graphed in Fig. 1. The steady-state levels of selected envelope glycoproteins in the lysates and supernatants of transfected COS-1 cells are shown in Fig. 2. For the wild-type envelope glycoproteins, the gpl20 molecule was found in both cell lysates and supernatants, while 1-20 NOTES 2121 the gpl60 and gp4l molecules were found only in the cell lysates (Fig. 2A, lanes 2 and 5). For several of the mutants, the levels and distribution of gpl60 and gpl20 glycoproteins were similar to those seen for the wild-type envelope glycoproteins. A small number of mutations resulted in gpl60 precursor envelope glycoproteins that were not processed to mature envelope glycoproteins (17). For these mutants, only the gpl60 glycoprotein was evident in the transfected COS-1 cell lysates and no gpl20 glycoprotein was detectable in either cell lysates or supernatants (data not shown) (17). Nine of the mutations resulted in a phenotype consistent with nearly complete loss of association of the gpl20 and gp4l envelope subunits. For these mutants, the predominant cell-associated envelope glycoprotein was gpl60, with little gpl20 detectable in the transfected cell lysates. The gp120 glycoprotein was present at wild-type levels or, in some cases, at higher than wild-type levels in transfected-cell supernatants (Fig. 2A and B). The cell-associated levels of the gp4l transmembrane envelope glycoprotein were decreased for these mutants relative to those seen for the wild-type envelope glycoprotein (Fig. 2A, lane 3). This suggests that the gp4l transmembrane glycoprotein is rapidly degraded following loss of association with gpl20. Two regions located near the termini of the mature gp120 glycoprotein appear to be particularly sensitive to change with respect to subunit association. The amino-terminal region (residues 36 to 45) consists primarily of hydrophobic amino acids that are highly conserved among the primate immunodeficiency viruses (Fig. 3). The carboxy-terminal region (residues 491 to 501) is also highly conserved but less hydrophobic than the amino-terminal region. It is noteworthy that even relatively conservative substitutions in either of these regions (e.g., valine 36 to leucine or isoleucine 491 to phenylalanine) can apparently disrupt gpl2o-gp4l interactions. Some gpl20 mutants exhibited a ratio of cell-associated gp120 to supernatant gpl20 that was lower than that seen for the wild-type virus. These changes may affect the affinity of gpl20 association with the gp4l transmembrane glycoprotein without causing a complete dissociation of the envelope subunits. This phenotype was observed for some mutations affecting the third (C3) and fourth (C4) conserved regions of the gpl20 glycoprotein. The C3 region (residues 380 to 384) consists of a stretch of well-conserved, hydrophobic amino acids located between two cysteine residues. These cysteine residues form disulfide bonds with cysteines flanking the C4 region (13), suggesting that these two regions are proximal on the native gpl20 molecule. The C4 amino acids that influence the interaction of gpl20 with gp4l are located in two hydrophobic, well-conserved stretches (residues 420 to 427 and 433 to 438). For the gpl20 mutants exhibiting decreased association indices, the degree of processing of the gpl60 envelope glycoprotein precursor and the CD4-binding ability of the mature gpl20 glycoprotein are listed in Table 2. These values FIG. 1. Location of amino acid changes affecting gpl2o-gp4l association. The HIV-1 gp120 exterior envelope glycoprotein is shown, with numbers representing amino acid residues. The regions that are relatively conserved (Cl to C5) in primate immunodeficiency viruses are shown in open bars, while the regions of variability (Vi to V5) are shaded. Darker shading represents greater variability. The signal sequence is designated S. The negative log of the association index is plotted on the vertical axis. For mutants indicated by a black vertical bar, both gpl60 precursor processing and CD4 binding are at least 40% of the wild-type values. For mutants indicated by stippled bars, gp160 precursor processing is less than 40% that of the wild-type glycoproteins. For mutants indicated by open bars, the association index, processing index, and CD4-binding ability are all less than 40% of wild-type levels. The open triangles indicate the positions of amino acids at which changes significantly affect CD4 binding. The solid and dotted circles are equivalent to the black and stippled bars, respectively.

4 2122 NOTES J. VIROL. Cl region HIV-1 L W V T V Y Y G VP VW K ()A HIV-2 q Y V T V F Y G v P (v) W(') N A SIvagm W I T V F Y G I P V W K N S SIVMND Q Y V T V F Y G V P V W K E A C5 region HIV-1 E L Y K Y K V ()HEp LG(i) A P T()A K R R V V(q)R E K R HIV-2 E L G D Y K L v E ( T P I G(f) A P T () v K R Y... SIVagm E L G R Y K L V E I T P I G F A P T E V R R Y... SIVMND Y G A H Y K LV K I M P I G I A P T D V R R H... FIG. 3. Predicted amino acid sequence of gpl20 regions in which several changes resulted in nearly complete loss of gpl2o-gp4l association. Capital letters indicate amino acids that are unchanged in different HIV-1 and HIV-2 isolates, whereas lowercase letters indicate residues that are found in most but not all HIV-1 and HIV-2 isolates. The residues that are boxed are identical or exhibit strong conservation of features in primate immunodeficiency viruses. The numbers indicate the residues at which changes resulted in significant reductions in gpl20 association with the expressing cell. The solid arrowhead designates the position of the proteolytic cleavage between the gpl20 and gp4l glycoproteins. SIVMND represents the sequence of a mandrill SIV (16). TABLE 2. Characterization of selected gp120 mutants aid in assessing the effect of the mutation on the overall conformation of the gpl20 glycoprotein. For four of the mutants (256 S/Y, 262 N/T, 447 S/I, and 482/483/484 ELY/ GRA), significant decreases in precursor processing and CD4 binding suggest that the amino acid changes resulted in global disruption of gpl20 conformation. (Mutants are designated by amino acid position and change; e.g., 256 S/Y has the S at position 256 changed to Y.) Other mutants exhibited decreased gpl60 processing but intact CD4-binding ability, suggesting that conformational effects of the amino acid change delayed the folding and processing of the precursor but that the processed fraction achieves an approximately correct conformation. Some of the changes resulting in almost complete dissociation of gpl20 from gp4l (e.g., 36 V/L, 40 Y/D, 491 I/F, 495 G/K, and 500/501 KA/KGIPKA) and most of the changes in the C3 and C4 regions resulting in partial dissociation did not significantly affect either precursor processing or CD4 binding. These results suggest that these changes are not causing loss of gpl20 cell association merely by global disruption of conformation. These results implicate two regions at the amino and carboxyl termini of the gpl20 molecule in the association with the gp4l transmembrane glycoprotein. These two regions represent the most highly conserved stretches of gpl20 amino acids when different primate immunodeficiency viruses are compared. Our results are consistent with models in which these two regions interact directly with the highly conserved exterior domain of gp4l or interact with other gpl20 sequences (perhaps with each other) to form a gpl20 molecule efficient at binding gp4l. With respect to the former model, it is noteworthy that the interaction of the influenza virus hemagglutinin HA1 receptor-binding glycoprotein with the HA2 transmembrane glycoprotein involves both aminoand carboxy-terminal sequences of HA1, although this interaction is stabilized by a disulfide bond (22). These studies also suggest that some hydrophobic sequences in the C3 and C4 regions may influence the interaction of gpl20 with gp4l. These sequences lie adjacent to residues implicated in CD4 binding (17) (Fig. 3). This relationship may be important if CD4 binding triggers changes in Mutant Association Processing Relative CD4 indexa index' bindingc Wild type V/L Y/D W/S P/Y N/P Q/F R/W S/Y N/T G/F /381 GE/YV E/P F/L Y/E K/L W/V < W/S < A/L Y/H Y/S P/R S/I P/L P/G D/S /483/484 ELY/GRA < I/F P/K G/K /498/499 APT/VLL < /501 KA/KGIPKA a Association indices were obtained from Table 1. b The processing index is a measure of the conversion of mutant gpl60 envelope glycoprotein precursor to mature gpl20 relative to that of the wild-type glycoprotein. Transfected COS-1 cells were continuously labeled with [35S]cysteine for 12 h, and cell lysates and supernatants were immunoprecipitated with excess serum (19501) from an AIDS patient. The amounts of gpl60 and gpl20 glycoproteins were determined by densitometric scanning of autoradiograms of sodium dodecyl sulfate-polyacrylamide gels. The processing index was calculated by the formula: processing index = ([total gp12o].,a., x [gpl6o]wi,d-,ype)/([gpl6o]muta,, x [total gpl2olwi1d type). ' The values for relative CD4 binding ability were reported in reference 17.

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