Changes in the Transmembrane Region of the ... - Journal of Virology

Vol. 64, No. 12

JOURNAL OF VIROLOGY, Dec. 1990, p. 6314-6318

0022-538X/90/126314-05$02.00/0 Copyright © 1990, American Society for Microbiology

Changes in the Transmembrane Region of the Human Immunodeficiency Virus Type 1 gp4l Envelope Glycoprotein Affect Membrane Fusion EIRIK HELSETH,l UDY OLSHEVSKY,l DANA GABUZDA,1 BLAIR ARDMAN,2 WILLIAM HASELTINE,3 AND JOSEPH SODROSKI1* Division of Human Retrovirology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School,1 and Department of Cancer Biology, Harvard School of Public Health,3 44 Binney Street, Boston, Massachusetts 02115, and Department of Medicine, Division of Hematology-Oncology, New England Medical Center Hospitals, Boston, Massachusetts 021112 Received 15 December 1989/Accepted 6 September 1990 The charged amino acids near or within the membrane-spanning region of the human immunodeficiency virus type 1 gp4l envelope glycoprotein were altered. Two mutants were defective for syncytium formation and virus replication even though levels of envelope glycoproteins on the cell or virion surface and CD4 binding were comparable to those of the wild-type proteins. Thus, in addition to anchoring the envelope glycoproteins, sequences proximal to the membrane-spanning gp4l region are important for the membrane fusion process.

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immune deficiency syndrome (AIDS) (1, 6, 19), which is characterized by depletion of CD4-positive lymphocytes (8, 16). The tropism of HIV-1 for CD4-positive cells is due to a specific interaction between CD4, the viral receptor, and the gpl20 exterior envelope glycoprotein (4, 11, 12, 18). After receptor binding, the viral envelope glycoproteins gp120 and gp4l mediate the fusion of the viral and host cell membranes to allow viral entry (24). The HIV-1 envelope glycoproteins expressed on the surface of the infected cell mediate the formation of multinucleated giant cells or syncytia (17, 22). Syncytium formation requires proteolytic processing of the gp160 envelope precursor, cell surface expression, association of the gp120 and gp4l subunits, CD4 binding, and membrane fusion events that follow CD4 binding (13). Mutations affecting the hydrophobic gp4l amino terminus abrogate membrane fusion events and can affect virus entry and/or syncytium formation (5, 7, 13). The HIV-1 gp4l transmembrane envelope glycoprotein contains a second hydrophobic region that stops translocation through the lipid bilayer of the endoplasmic reticulum and serves to anchor the envelope glycoproteins in the membrane. Deletion of this region results in the production of soluble envelope glycoproteins (2, 13). While the membrane-spanning regions of most viral envelope proteins consist of stretches of hydrophobic amino acids, the membraneproximal regions of most lentivirus transmembrane glycoproteins are interrupted by charged residues (10, 21, 23). For example, while HIV-1 has two charged residues (at positions 707 and 709) carboxyl to the membrane-spanning region, there are two basic residues (lysine 683 and arginine 696) that are located within the hydrophobic segment encompassing the transmembrane region. Each of the four positively charged amino acids proximal to the HIV-1 gp4l membrane-spanning region was changed, by site-directed mutagenesis (15), to examine the effect of such alterations on envelope glycoprotein structure and *

function (Fig. 1). A fifth mutant was unintentionally generated, resulting in an insertion of three additional amino acids into the gp4l transmembrane region and the conversion of arginine 696 to valine. The mutated env genes were introduced into the pSVIIIenv plasmid, which allows transient expression in transfected COS-1 and Jurkat-tat cells (3, 9, 13, 20). Transfected cells were radiolabeled with [35S]CySteine, and steady-state levels of envelope glycoprotein expression were assessed by precipitation of cell lysates and supernatants with serum (RV119) from a patient with AIDS as described previously (9, 13). The pattern and level of proteins precipitated from COS-1 cell lysates and supernatants were similar for wild-type and all five mutant envelope glycoproteins (Fig. 2). By contrast, in Jurkat-tat cells, both the proteolytic processing and the steady-state levels of the 683 K/I and the 696 RIVGLS mutants were decreased compared with those of the wild-type protein. The processing and levels of expression of the other three mutants did not differ from those of the wild-type protein in Jurkat-tat cells (data not shown). To examine whether the 683 K/I and 696 R/VGLS mutations affected the stability of the envelope glycoproteins expressed in COS-1 cells, a pulse-chase analysis of transfected cells was performed. No difference in the processing or stability of these mutants was observed relative to those of the wild-type envelope glycoproteins (data not shown). The presence of wild-type levels of gpl20 glycoprotein in the supernatants of COS-1 cells transfected with the plasmids expressing the mutant envelope glycoproteins suggested that these mutant envelope proteins were expressed on the cell surface. To examine this directly, metabolically labeled COS-1 cells were incubated with antiserum from a patient with AIDS and unbound antibody was removed by thorough washing. The cells were then lysed and immune complexes were precipitated by binding to protein A-Sepharose beads. The precipitates were analyzed on sodium dodecyl sulfate-polyacrylamide gels to determine the level of envelope glycoproteins accessible to the antibody. The cell surface-accessible amount of envelope glycoproteins was comparable for the wild-type and mutant envelope proteins (Fig. 3).

Corresponding author. 6314

VOL. 64, 1990

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To examine cell surface expression of the mutant envelope glycoproteins by another method, transfected COS-1 cells were iodinated by the lactoperoxidase method (14). Cell lysates were precipitated with serum from a patient with AIDS (Fig. 4). The wild-type and mutant gpl20 envelope glycoproteins and, to a lesser extent, the gpl60 envelope glycoproteins were iodinated, indicating that these proteins were expressed on the cell surface. .Since the processing, steady-state level, and cell surface expression of all five mutant envelope glycoproteins were comparable in COS-1 cells, the function of the proteins was assessed in this cell type. To assess the ability of the mutant glycoproteins to mediate the formation of syncytia, COS-1 cells transfected with the envelope-expressing plasmids were cocultivated with CD4-positive SupTl lymphocytes. Following overnight incubation, syncytia were scored. The syncytium-forming abilities of the 683 K/I and the 696 R/VGLS mutants were significantly reduced compared with that of the wild-type glycoproteins (Table 1). The other mutants exhibited significant syncytium-forming activity, although syncytium formation induced by the 707 RII mutant was reduced to 65% of that of the wild-type glycoproteins. One potential reason for the decreased syncytium-forming ability of mutant envelope glycoproteins is a reduction in ability of the gpl20 glycoprotein to bind CD4. To measure CD4 binding of the mutants, metabolically labeled supernatants from transfected COS-1 cells, which contain free gpl20 because of the lability of gp120-gp41 association, were incubated with SupTl human T lymphocytes, which were then pelleted by centrifugation, washed, and lysed. Bound gpl20 was measured by immunoprecipitation of the lysed target cell. The results shown in Fig. 2 indicate that no

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FIG. 1. Mutations affecting the gp4l transmembrane region. A diagram of the HIV-1 envelope glycoproteins is shown, with the positions of the amino acids changed by the mutations marked. The extent of the hydrophobic stretch surrounding the membranespanning region is designated TM. An uninterrupted amino acid sequence for this region is provided. Charged amino acids within this sequence that have been changed are circled. The mutations and the effects on the amino acid sequence of the gp4l glycoprotein are listed.

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FIG. 2. Structure of the mutant envelope glycoproteins expressed in COS-1 cells. Radioimmunoprecipitates from cell lysates (A) or supernatants (B) of COS-1 cells either mock transfected (lanes 1) or transfected with pSVIIIenv (lanes 2), pSVIIIenv-683 K/I (lanes 3), pSVIIIenv-696 RNVGLS (lanes 4), pSVIIIenv-707 R/I (lanes 5), or pSVIIIenv-709 R/L (lanes 6) DNA are shown. The immunoprecipitates of labeled gpl20 bound to SupTl CD4-positive lymphocytes are shown (C), with lane numbers representing the same plasmids used for gpl20 production in COS-1 cells as for panels A and B. The positions of gpl60, gpl20, and gp4l glycoproteins are indicated. The results obtained for the COS-1 cells transfected with the pSVIIIenv-696 R/S plasmid were similar to that obtained following transfection with the pSVIIIenv

plasmid (data not shown). Molecular masses (in kilodaltons) are indicated.

6316

NOTES

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FIG. 4. Cell surface envelope glycoproteins as detected by iodination. Immunoprecipitation of iodinated COS-1 cells transfected with no DNA (lane 1) or with pSVIIIenv (lane 2), pSVIIIenv-683 K/I (lane 3), pSVIIIenv-696 R/VGLS (lane 4), pSVIIIenv-696 R/S (lane 5), pSVIIIenv-707 R/I (lane 6), or pSVIIIenv-709 R/L (lane 7) plasmid DNA is shown. The positions of gpl60 and gpl20 are indicated. The slight reduction in iodinated gpl20 for the pSVIIIenv707 R/I and pSVIIIenv-709 R/L plasmids seen in this experiment was not reproducible.

41

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FIG. 3. Cell surface-accessible envelope glycoproteins. The amounts of envelope glycoprotein accessible to antibody incubated

with intact COS-1 cells transfected with no DNA (lane 1) or with pSVIIIenv (lane 2), pSVIIIenv-683 K/I (lane 3), pSVIIIenv-696 R/VGLS (lane 4), pSVIIIenv-707 R/I (lane 5), or pSVIIIenv-709 R/L (lane 6) plasmid DNA are shown. The results obtained for the COS-1 cells transfected with the pSVIIIenv-696 R/S plasmid were similar to that obtained following transfection with the pSVIIIenv plasmid (data not shown). Molecular masses (in kilodaltons) are indicated on the right.

difference in CD4-binding ability was detected among the mutant and wild-type envelope glycoproteins. To assess the ability of the mutant envelope glycoproteins to support cell-free virus transmission, a transient envelope complementation assay (9) was employed. The envelopeexpressing plasmid was cotransfected into COS-1 cells with the pHXBAenvCAT plasmid. The latter plasmid contains an HIV-1 provirus with a large deletion in the env gene and expresses the bacterial chloramphenicol acetyltransferase (CAT) gene, which replaces the nef gene. The ability of the recombinant virions produced in the transfected COS-1 cell supernatants to enter T lymphocytes is dependent upon the expression of replication-competent HIV-1 envelope glycoproteins (9). The efficiency of this single round of cell-free virus entry was assessed by incubating the filtered virions with Jurkat lymphocytes and measuring CAT activity in the latter cells as described elsewhere (9). Cell-free virus transmission supported by the 683 K/I and 696 R/VGLS mutants was significantly attenuated relative to that of the wild-type

envelope glycoproteins (Table 1). Some reduction in cellfree transmission was associated with the 696 R/S, 707 RII, and 709 R/L changes. The reduction in virus transmission observed for the 683 R/I and 696 R/VGLS mutants was not due to changes in the level of virion-associated envelope glycoproteins. [35S]cysteine-labeled supernatants of COS-1 cells cotransfected with the envelope-expressing plasmid and the pHXBAenvCAT plasmid were centrifuged at low speed (800 x g) to clear cell debris and then centrifuged at 12,000 x g to pellet virions. Both virion pellets and supernatants were lysed and immunoprecipitated as described previously (13). The amount of virion-associated gp120 glycoprotein relative to that of core proteins was the same for mutant and wild-type glycoproteins (Fig. 5). The presence of charged amino acids within or near the membrane-spanning segments of lentivirus envelope glycoproteins suggests an unusual structure for these regions. The work described herein indicates that some changes in the hydrophobic HIV-1 gp4l region proximal to the transmembrane region disrupt the ability of the envelope glycoproteins TABLE 1. Syncytium formation and replication complementation of HIV-1 gp4l mutants expressed in COS-1 cells Mutant

%formationa Syncytium

% Replication b complementation

None (wild type) 683 K/I 696 R/S 696 R/VGLS 707 R/I 709 R/L

100 5 87 14 65 84

100 0 74 0 24 40

a Values represent the number of syncytia formed upon cocultivation of COS-1 cells expressing the mutant envelope glycoproteins with SupTl cells, relative to that observed for the wild-type proteins. b Values represent the CAT conversion in target Jurkat cells for the mutant envelope glycoproteins relative to the value for the wild-type glycoproteins.

NOTES

VOL. 64, 1990

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10. FIG. 5. Virion-associated envelope glycoproteins. Immunoprecipitates of supernatant fractions of COS-1 cells transfected with the pHXBAenvCAT plasmid alone (lanes 1) or with pHXBVAenvCAT plus either pSVIIIenv (lanes 2), pSVIIIenv-683 K/I (lanes 3), or pSVIIIenv-696 RNVGLS (lanes 4) are shown. Virion pellets derived from transfected COS-1 cell supernatants (A) and the supernatant fraction remaining after the virions had been pelleted by centrifugation (B) are shown.

to form syncytia and to complement virus entry,

11.

12.

even

though processing, subunit association, cell or virion surface expression, and CD4 binding were not apparently affected. Thus, this region may be directly or indirectly involved in membrane fusion events, in addition to membrane anchorage. The process of membrane fusion initiated by gpl2O-CD4 binding is apparently dependent on the structural integrity of both hydrophobic gp4l segments, the amino-terminal domain, and the transmembrane domain. These results are consistent with models of membrane fusion in which these hydrophobic sequences interact with each other or with the lipid membranes to be fused or both. We thank Robert Gallo, Flossie Wong-Staal, Max Essex, and Bruce Walker for reagents; Jan Welch for manuscript preparation; and Amy Emmert for artwork. This work was supported by a fellowship from the Norwegian Cancer Society (to E.H.), by a grant from the Leukemia Society of America (to J.S.), by a grant from the John Hartford Foundation (to B.A.), and by Public Health Service grants AI24755 and A127729 from the National Institutes of Health. LITERATURE CITED 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-

13.

14.

15. 16.

17.

18.

6317

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