The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells.

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5'GCGCGC3') or an arbitrary (5'ACGCGT3') palindrome sequence in place of the 39-nucleotide DIS stem-loop (NL(CGCGCG) and NL(ACGCGT)). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

Evidence that the transmembrane domain proximal region of the human T-cell leukemia virus type 1 fusion glycoprotein gp21 has distinct roles in the prefusion and fusion-activated states.

To investigate the structural context of the fusion peptide region in human T-cell leukemia virus type 1 gp21, maltose-binding protein (MBP) was used as an N-terminal solubilization partner for the entire gp21 ectodomain (residues 313-445) and C-terminally truncated ectodomain fragments. The bacterial expression of the MBP/gp21 chimeras resulted in soluble trimers containing intramonomer disulfide bonds. Detergents blocked the proteolytic cleavage of fusion peptide residues in the MBP/gp21-(313-425) chimera, indicating that the fusion peptide is available for interaction with detergent despite the presence of an N-terminal MBP domain. Limited proteolysis experiments indicated that the transmembrane domain proximal sequence Thr(425)-Ala(439) protects fusion peptide residues from chymotrypsin. MBP/gp21 chimera stability therefore depends on a functional interaction between N-terminal and transmembrane domain proximal regions in a gp21 helical hairpin structure. In addition, thermal aggregation experiments indicated that the Thr(425)-Ser(436) sequence confers stability to the fusion peptide-containing MBP/gp21 chimeras. The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity. By contrast, single alanine substitutions in MBP/gp21 did not significantly alter chimera stability, indicating that multiple residues within the transmembrane domain proximal region and the fusion peptide and adjacent glycine-rich segment contribute to stability, thereby mitigating the potential effects of the substitutions. The fusion-enhancing effects of the substitutions are therefore likely to be caused by alteration of the prefusion complex. Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21.

Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association.

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure.

Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins.

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-A resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Evolutionary conservation of the membrane fusion machine.

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Efficacy of fusion peptide homologs in blocking cell lysis and HIV-induced fusion.

Contrary to earlier reports, we have found that tri- and hexapeptides analogous or homologous with segments of the 23-residue N-terminal fusion sequence (FS) of the viral transmembrane glycoprotein gp41 (residues 517-539) did not significantly inhibit HIV-1-induced syncytium formation, using an uninfected cell-infected cell fusion assay. In contrast, we found that the high molecular weight apolipoprotein A-1 and a 23-residue analog of the FS, with the phenylalanine residues at positions 524 and 527 replaced with alanine residues, were effective inhibitors. Although the tripeptides were ineffective as inhibitors of syncytium formation, we found a number of them inhibited red cell lysis induced by the synthetic peptide AVGIGALFLGFLGAAGSTMGARS (based on the HIV-1 gp41 FS). This effect was also seen with apolipoprotein A-1. The Ala524,527 analog of the fusion sequence could not be tested in this system because it was hemolytic. We concluded that the smaller peptides were effective inhibitors of hemolysis because they interfered with pore formation by the fusion sequence peptide, either by disrupting the pores or by preventing the peptide from adopting the alpha-helical conformation found in the pores. On the other hand, membrane fusion, which is a prelude to syncytium formation, has been shown to require the fusion sequence in the beta-strand conformation. We argue that small peptides would be unable to block interaction between such strands, although larger molecules, such as apolipoprotein A-1 and the Ala524,527 analog, would be able to do so and thus inhibit fusion. It seems, therefore, that a successful drug directed against the FS-cell membrane interaction stage of syncytium formation would need to be of relatively high molecular weight and complexity.

Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers.

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic alpha-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.

Determinants of human immunodeficiency virus type 1 envelope glycoprotein oligomeric structure.

Oligomerization of the human immunodeficiency virus type 1 envelope (env) glycoproteins is mediated by the ectodomain of the transmembrane glycoprotein gp41. We report that deletion of gp41 residues 550 to 561 resulted in gp41 sedimenting as a monomer in sucrose gradients, while the gp160 precursor sedimented as a mixture of monomers and oligomers. Deletion of the nearby residues 571 to 582 did not affect the oligomeric structure of gp41 or gp160, but deletion of both sequences resulted in monomeric gp41 and predominantly monomeric gp160. Deletion of residues 655 to 665, adjacent to the membrane-spanning sequence, partially dissociated the gp41 oligomer while not affecting the gp160 oligomeric structure. In contrast, deletion of residues 510 to 518 from the fusogenic hydrophobic N terminus of gp41 did not affect the env glycoprotein oligomeric structure. Even though the mutant gp160 and gp120 molecules were competent to bind CD4, the mutations impaired fusion function, gp41-gp120 association, and gp160 processing. Furthermore, deletion of residues 550 to 561 or 550 to 561 plus 571 to 582 modified the antigenic properties of the proximal residues 586 to 588 and the distal residues 634 to 664. Our results indicate that residues 550 to 561 are essential for maintaining the gp41 oligomeric structure but that this sequence and additional sequences contribute to the maintenance of gp160 oligomers. Residues 550 to 561 map to the N terminus of a putative amphipathic alpha-helix (residues 550 to 582), whereas residues 571 to 582 map to the C terminus of this sequence.

Synthetic peptides representing sequences within gp41 of HIV as immunogens for murine T- and B-cell responses.

Within the gp41 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) there is a relatively conserved region which appears accessible to the immune system during the course of HIV infection and is recognised by antibody from virtually all patients with AIDS. This region has also been shown to function as a target for human T cells. We have examined synthetic peptides spanning this sequence, between residues 572 and 604, with a view to evaluating their potential as immunogens. Peptides 572GIKQLQARILAVERYLKDQQ591 and 579RILAVERYLKDQQLLGGIWGCSGK601 were good immunogens in two different strains of mice while peptide 576LQARILAVERYLKDQQ591 was an inferior immunogen, and peptide 593LGIWGCSGKLIC604 was non-immunogenic unless coupled to a carrier protein. For both antibody and T cell responses it was apparent that sequences that could function as determinants within one peptide could not do so in the context of a different peptide immunogen. It follows that by judicious choice of immunogen sequence it may be possible to direct the immune response towards a desired fine specificity. Unwanted responses by CD4+ T cells isolated from certain peptide-primed animals were also observed. These T cells showed an unusual reactivity in that they were incapable of recognising their determinant AVERYLKDQQ if it was extended at the C-terminal end with the native sequence and as such would not be expected to recognise the native molecule unless processing created the identical C-terminus.

Studies on the antibody response of mice and humans after immunization with potential influenza virus A (H1N1) vaccines.

The antibody response of mice and adult humans to immunization with subunit vaccines derived from a pair of antigenically distinct influenza A H1N1 viruses isolated in eggs was investigated. Although the haemagglutinin molecule of each virus differed by only three amino acid residues, highly specific antibody responses were elicited in mice as determined by haemagglutination inhibition and radioimmunoprecipitation assays. Results from competitive radioimmunoassays using monoclonal antibodies of known specificity and a study of the reactivity of mouse antisera with H1N1 field strains indicated that the marked differences in the antibody responses to the two vaccines was due to an amino acid substitution in the distal tip of the haemagglutinin molecule. In contrast, crossreactive antibody responses were elicited in humans presumably due to exposure to viruses related to the candidate vaccine prior to vaccination. Although immunogenic differences are apparent in this pair of antigenically distinct viruses in naive laboratory animals, these differences are not apparent following vaccination of humans that had prior exposure to related viruses.

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.

N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.

Three antibody molecules can bind simultaneously to each monomer of the tetramer of influenza virus neuraminidase and the trimer of influenza virus hemagglutinin.

Trimeric hemagglutinin and tetrameric neuraminidase molecules isolated from influenza virus bind an average of 9 and 13 molecules respectively of monovalent antibody fragments prepared from IgG isolated from polyclonal sera. In each case this represents an average of approximately three molecules of antibody binding to each protomer. Although there is compelling evidence for the presence of multiple adjacent and overlapping epitopes covering the surface of these two viral antigens, steric hindrance ensures that even under saturating conditions only three molecules of monovalent antibody fragments can be simultaneously accommodated on each monomer.

The stoichiometry of binding between monoclonal antibody molecules and the hemagglutinin of influenza virus.

The number of neutralizing monoclonal IgG molecules that can bind to a single trimeric molecule of influenza viral hemagglutinin (HA) was calculated by estimating the molecular weight of the immune complexes formed under conditions of antibody excess and was found to be dependent upon the antigenic site to which the MAb is directed. Whereas three antibody molecules directed to site A or site E are able to bind simultaneously to a single trimer of HA, generally only one molecule directed to site B (the "tip") or site B/D ("tip/interface") can be accommodated. Using mixtures of MAbs, more IgG molecules can be accommodated, but steric hindrance limits simultaneous binding of different MAbs directed to the same antigenic site or even to neighboring sites. At limiting antibody concentration, some MAbs can form much larger aggregates in which several HA molecules are crosslinked by antibody. However, the fact that certain MAbs do not crosslink HA molecules in this way indicates that MAbs directed to different epitopes within the same general antigenic site differ significantly in their geometry of binding.

Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three amino acid residues.

Two monoclonal antibodies recognizing distinct epitopes the outer boundaries of which are separated by only three amino acid residues, a maximum of 10A, were demonstrated to bind simultaneously to a short synthetic peptide. The affinity of binding of the two monoclonal antibodies and of Fab' fragments derived from them was determined. The stoichiometry of the interaction was analysed by velocity sedimentation and by gel permeation chromatography experiments. The results indicate that the immune complexes formed are composed of two antibody molecules in association with one or two peptide molecules.

Direct role of viral hemagglutinin in B-cell mitogenesis by influenza viruses.

The mitogenic activity of influenza virus is a function of the hemagglutinin (HA) molecule. Purified HA is mitogenic for murine B lymphocytes but not T lymphocytes. Furthermore, like the intact virus, HA of the H2 (but not H3) subtype is mitogenic only for B cells expressing the class II major histocompatibility complex glycoprotein I-E. Since virus bearing uncleaved HA is as mitogenic as virus bearing cleaved HA, the membrane fusion activity of the HA molecule is not involved.