AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 9, 1992 Mary Ann I .¡chert. Inc., Publishers
Studies
the Role of the V3 Loop in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Function
on
SHIOW-HER
CHIOU,1
ERIC O.
FREED,2'3 ANTONITO T. PANGANIBAN,2 and WILLIAM
R.
KENEALY1
ABSTRACT Mutations within the principal neutralizing determinant (the V3 loop) of the HIV-1 surface envelope glycoprotein gpl20 block or greatly reduce the ability of the HIV-1 envelope glycoprotein to induce cell fusion in CD4+ HeLa T4 cells while keeping its CD4 binding ability. However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4. To investigate the role that the V3 loop plays in gp 160 processing and CD4 binding, we deleted the entire V3 loop region of the HIV-1 env gene. The resultant glycoprotein could not mediate cell fusion in the HeLa T4 cell line and no proteolytic processing of gp 160 or CD4 binding could be detected. To test whether any domain of the V3 loop is involved in attaining the proper envelope glycoprotein conformation required for proteolytic processing and CD4 binding, we introduced a series of deletions into the coding region of the V3 loop. Most of the residues within the V3 loop could be removed while retaining gpl60 processing and CD4 binding. Our results indicate that the cysteines that form the V3 loop or the disulfide bond itself are important for proper envelope glycoprotein folding and processing. Because many of the mutants constructed in this study do not contain the type-specific neutralizing determinant of HIV-1, they may be potential reagents to bind group-specific neutralizing antibodies or to elicit a group-specific neutralizing response against HIV-1.
INTRODUCTION
The
human immunodeficiency virus
disulfide bridge between two invariant cysteine residues at gpl 20 amino acid 303 and 338.7il This loop is comprised of the third hypervariable region of gpl 20, and is therefore referred to as the V3 loop.7'9 Most of the amino acids within the V3 loop are hypervariable between different strains of HIV-1. However, a highly conserved G-P-G-R motif is located in the middle of the V3 loop.I0 Residues in the left and right stem of the loop are also highly conserved.10 Neutralizing antibodies to the region en¬ compassing the highly conserved tip can block HIV-1 infection and inhibit cell fusion in a type-specific manner, without ' ' preventing gpl20 binding to CD4. A five amino acid linker insertion mutation in the V3 loop resulted in a glycoprotein which failed to form syncytia in transfected CD4+ cells, but still bound CD4.12 Recent studies indicated that mutations in the highly conserved tip of the loop or at highly conserved residues in the stems of the loop either a
type 1 (HIV-1)
en¬
codes a precursor envelope glycoprotein gpl60 which is cleaved by a host protease to form the external envelope glycoprotein gpl20 and the transmembrane envelope glycopro¬ tein gp41.'~3 The gpl20 on the viral envelope binds to cells bearing the HIV receptor molecule CD4 and, acting in concert with gp41, leads to the fusion of the viral envelope with the host cell membrane.4,5 In addition to infecting as a cell-free virion, HIV can be passed from cell to cell by fusion of an infected cell with an uninfected CD4-bearing cell. The process of cell fusion leads to the formation of multinucleated giant cells called syncytia.5·6 After infection, most patients produce specific antibodies directed against HIV-1. The principal neutralizing determinant (PND) of HIV-1 is located within a loop formed by
'Department of Biochemistry and 2McArdle Laboratory for Cancer Research University of Wisconsin-Madison, Madison, WI 53706. 'Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Building 4, National Institutes of Health, Bethesda, Md 20892. 1611
CHIOU ET AL.
1612
completely or reduced greatly the ability of the HIV-1 envelope glycoprotein to induce cell fusion without influencing envelope glycoprotein synthesis, processing, transport or bind¬ ing to the CD4 receptor molecule.9'3'4 However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4.9'5 Elim¬ ination of the V3 disulfide linkage may thus greatly alter the conformation of envelope glycoprotein such that many of its functions are impaired. We deleted the entire V3 loop region of the HIV-1 env gene to '6 test this hypothesis, using pHenv9 transfected HeLa T4 cells as a model system. pHenv is an HIV-1 envelope glycoprotein expression vector which can efficiently express envelope glyco¬ proteins when transfected into the CD4+ HeLa cell line HeLa blocked
T4.9 This cell line has been used by several groups for cell fusion
studies because it can grow adherently and form easily quantifi¬ able syncytia.9'5·'6'8 Similar to cell lines,19 this cell line is infectable by HIV17 and has proven useful for envelope glyco¬
protein studies.913 Because point mutations within the V3 loop of gpl 20 primar¬ ily influence its ability to mediate cell fusion,9'3'4 it is plausible that the deletion of a portion of the V3 loop might not affect gp 160 processing or CD4 binding to any dramatic degree. To test this hypothesis and to determine whether any domain of the V3 loop is required for proper glycoprotein folding, we introduced a series of partial V3 loop deletions into pHenv9 and analyzed the ability of mutant envelope glycoproteins to induce cell fusion, to undergo proteolytic processing and to bind CD4 in
resulted in 470-570 bp BgHI fragments containing the desired mutations. The 470-570 bp BgHI fragments containing a series of partial V3 loop deletions were then introduced into pHenv' to
generate pHen A319-322, pUen 324-328, pHen 312-321,
pHé7ívA323-335, and pHenvA306-322. The construction of pHenvA319-335 and pHenvA306-335 was done by exchanging the BssHII-Sal fragment of pHe«vA323-335 with that of pHcnvA319-322 and
Studies
the Role of the V3 Loop in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Function
on
SHIOW-HER
CHIOU,1
ERIC O.
FREED,2'3 ANTONITO T. PANGANIBAN,2 and WILLIAM
R.
KENEALY1
ABSTRACT Mutations within the principal neutralizing determinant (the V3 loop) of the HIV-1 surface envelope glycoprotein gpl20 block or greatly reduce the ability of the HIV-1 envelope glycoprotein to induce cell fusion in CD4+ HeLa T4 cells while keeping its CD4 binding ability. However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4. To investigate the role that the V3 loop plays in gp 160 processing and CD4 binding, we deleted the entire V3 loop region of the HIV-1 env gene. The resultant glycoprotein could not mediate cell fusion in the HeLa T4 cell line and no proteolytic processing of gp 160 or CD4 binding could be detected. To test whether any domain of the V3 loop is involved in attaining the proper envelope glycoprotein conformation required for proteolytic processing and CD4 binding, we introduced a series of deletions into the coding region of the V3 loop. Most of the residues within the V3 loop could be removed while retaining gpl60 processing and CD4 binding. Our results indicate that the cysteines that form the V3 loop or the disulfide bond itself are important for proper envelope glycoprotein folding and processing. Because many of the mutants constructed in this study do not contain the type-specific neutralizing determinant of HIV-1, they may be potential reagents to bind group-specific neutralizing antibodies or to elicit a group-specific neutralizing response against HIV-1.
INTRODUCTION
The
human immunodeficiency virus
disulfide bridge between two invariant cysteine residues at gpl 20 amino acid 303 and 338.7il This loop is comprised of the third hypervariable region of gpl 20, and is therefore referred to as the V3 loop.7'9 Most of the amino acids within the V3 loop are hypervariable between different strains of HIV-1. However, a highly conserved G-P-G-R motif is located in the middle of the V3 loop.I0 Residues in the left and right stem of the loop are also highly conserved.10 Neutralizing antibodies to the region en¬ compassing the highly conserved tip can block HIV-1 infection and inhibit cell fusion in a type-specific manner, without ' ' preventing gpl20 binding to CD4. A five amino acid linker insertion mutation in the V3 loop resulted in a glycoprotein which failed to form syncytia in transfected CD4+ cells, but still bound CD4.12 Recent studies indicated that mutations in the highly conserved tip of the loop or at highly conserved residues in the stems of the loop either a
type 1 (HIV-1)
en¬
codes a precursor envelope glycoprotein gpl60 which is cleaved by a host protease to form the external envelope glycoprotein gpl20 and the transmembrane envelope glycopro¬ tein gp41.'~3 The gpl20 on the viral envelope binds to cells bearing the HIV receptor molecule CD4 and, acting in concert with gp41, leads to the fusion of the viral envelope with the host cell membrane.4,5 In addition to infecting as a cell-free virion, HIV can be passed from cell to cell by fusion of an infected cell with an uninfected CD4-bearing cell. The process of cell fusion leads to the formation of multinucleated giant cells called syncytia.5·6 After infection, most patients produce specific antibodies directed against HIV-1. The principal neutralizing determinant (PND) of HIV-1 is located within a loop formed by
'Department of Biochemistry and 2McArdle Laboratory for Cancer Research University of Wisconsin-Madison, Madison, WI 53706. 'Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Building 4, National Institutes of Health, Bethesda, Md 20892. 1611
CHIOU ET AL.
1612
completely or reduced greatly the ability of the HIV-1 envelope glycoprotein to induce cell fusion without influencing envelope glycoprotein synthesis, processing, transport or bind¬ ing to the CD4 receptor molecule.9'3'4 However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4.9'5 Elim¬ ination of the V3 disulfide linkage may thus greatly alter the conformation of envelope glycoprotein such that many of its functions are impaired. We deleted the entire V3 loop region of the HIV-1 env gene to '6 test this hypothesis, using pHenv9 transfected HeLa T4 cells as a model system. pHenv is an HIV-1 envelope glycoprotein expression vector which can efficiently express envelope glyco¬ proteins when transfected into the CD4+ HeLa cell line HeLa blocked
T4.9 This cell line has been used by several groups for cell fusion
studies because it can grow adherently and form easily quantifi¬ able syncytia.9'5·'6'8 Similar to cell lines,19 this cell line is infectable by HIV17 and has proven useful for envelope glyco¬
protein studies.913 Because point mutations within the V3 loop of gpl 20 primar¬ ily influence its ability to mediate cell fusion,9'3'4 it is plausible that the deletion of a portion of the V3 loop might not affect gp 160 processing or CD4 binding to any dramatic degree. To test this hypothesis and to determine whether any domain of the V3 loop is required for proper glycoprotein folding, we introduced a series of partial V3 loop deletions into pHenv9 and analyzed the ability of mutant envelope glycoproteins to induce cell fusion, to undergo proteolytic processing and to bind CD4 in
resulted in 470-570 bp BgHI fragments containing the desired mutations. The 470-570 bp BgHI fragments containing a series of partial V3 loop deletions were then introduced into pHenv' to
generate pHen A319-322, pUen 324-328, pHen 312-321,
pHé7ívA323-335, and pHenvA306-322. The construction of pHenvA319-335 and pHenvA306-335 was done by exchanging the BssHII-Sal fragment of pHe«vA323-335 with that of pHcnvA319-322 and
