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PubMed2005

Mutations conferring resistance to human immunodeficiency virus type 1 fusion inhibitors are restricted by gp41 and Rev-responsive element functions.

One of the human immunodeficiency virus (HIV) envelope proteins, gp41, plays a key role in HIV fusion. A gp41-derived peptide, T-20, efficiently inhibits HIV fusion and is currently approved for treatment of HIV-infected individuals. Although resistant variants have been reported, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated variants resistant to C34, a peptide derived from the gp41 carboxyl terminus heptad repeat (C-HR) in vitro. The resistant variants had a 5-amino-acid deletion in gp120 and a total of seven amino acid substitutions in gp41. Binding assays revealed that an I37K substitution in the N-terminal heptad repeat (N-HR) impaired the binding of C34, whereas an N126K substitution in the C-HR enhanced the binding to mutated N-HR, indicating that both mutations were directly involved in resistance. On the other hand, substitutions for A30 and D36 seemed to be secondary mutations, located complementary to each other in the Rev-responsive element (RRE), and were mutated simultaneously to maintain the secondary structure of the RRE that was impaired by the mutations at I37. Thus, HIV acquired resistance to C34 by mutations in N-HR, which directly interacted with C34. However, since this region also encoded the RRE, additional mutations were required to maintain viral replication. These results suggest that HIV fusion is one of the attractive targets for HIV chemotherapy.

Amino Acid Sequence↗

PubMed2002

Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay.

In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit). HIV-1 gp41 subunit of subtype E was successfully expressed in E. coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C. The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively. The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity. The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA. The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht. This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand.

Base Sequence↗

PubMed2006

Arterial aneurysms in HIV patients: molecular mimicry versus direct infection?

There is growing literature on the subject of aneurysmal degeneration of arteries in patients who are infected with HIV. A patient recently seen at our medical center with an aneurysm of the carotid artery stimulated our interest in reviewing the mechanisms by which HIV may initiate or predispose to these pathologies. There are at least three major possibilities: (1) immunodeficiency allows bacteria that are known to cause mycotic aneurysms to proliferate without immune restraint; (2) one or more of the HIV envelope proteins sufficiently resemble one or more artery-specific-antigenic proteins (ASAPs) that may trigger an autoimmune response (molecular mimicry); and (3) the HIV virus itself infects arterial-resident cells that maintain the integrity of the load-bearing matrix. The computational searches reported here suggest that the ASAP, matrix cell adhesion molecule-1 (Mat-CAM-1), has a high degree of similarity to known ligands for HIV envelope proteins gp41 and gp120. No similarities of Mat-CAM-1 to the HIV envelope glycoproteins were detected. Accordingly, among the possibilities for explaining the HIV/aneurysm connection, direct infection of aortic fibroblasts by the HIV virus is more likely to be the pathogenetic mechanism than the process of molecular mimicry.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Analysis of the interaction between the HIV-inactivating protein cyanovirin-N and soluble forms of the envelope glycoproteins gp120 and gp41.

The novel virucidal protein cyanovirin-N (CV-N) binds with equally high affinity to soluble forms of either H9 cell-produced or recombinant glycosylated HIV-1 gp120 (sgp120) or gp160 (sgp160). Fluorescence polarization studies showed that CV-N is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41) (as well as SIV glycoprotein 32), albeit with considerably lower affinity than the sgp120/CV-N interaction. Pretreatment of CV-N with either sgp120 or sgp41 abrogated the neutralizing activity of CV-N against intact, infectious HIV-1 virions. Isothermal calorimetry and optical biosensor binding studies showed that CV-N bound to recombinant sgp120 with a K(d) value ranging from 2 to 45 nM and to sgp41 with a K(d) value of 606 nM; furthermore, they indicated an approximate 5:1 stoichiometry for CV-N binding to sgp120 and a 1:1 stoichiometry for CV-N binding to sgp41. Circular dichroism studies additionally illuminated the binding of CV-N with both sgp120 and sgp41, providing the first direct evidence that conformational changes are a consequence of CV-N interactions with both HIV-1 envelope glycoproteins.

Anti-HIV Agents↗

PubMed1994

The role of immunoglobulin binding factors in the pathogenesis and therapy of AIDS.

The human immunodeficiency virus (HIV) gp120 and gp41 envelope proteins and Staphylococcus aureus protein A (SPA) all have Fc receptor (FcR)-like immunoglobulin binding factor (IBF) activity for the Fc constant fragments of human immunoglobulin G (IgG). Viral IBF may contribute to the pathology of HIV by jamming the network of FcR signals that control FcR-dependent immunity. Conversely, the bacterial IBF SPA has anti-retroviral activity that may involve antagonism of the immunopathological action of viral IBF, strongly suggesting IBF may act as a double-edged sword that might be turned against viral invaders.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1.

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.

Chromatography, Affinity↗

PubMed2000

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia.

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.

AIDS Dementia Complex↗

PubMed1991

Identification of membrane anchorage domains of the HIV-1 gp160 envelope glycoprotein precursor.

The gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a hydrophobic membrane-spanning domain that serves to anchor the gp120-gp41 complex on the surface of infected cells and virions. To study the requirements for membrane anchorage, conservative amino acid substitutions in three residues at a time were made within this hydrophobic gp41 region. The complete gp160 precursor as well as the gp120 exterior envelope glycoprotein were exported into the supernatant of expressing cells for two mutants with amino acid substitutions in residues 687-689 and 697-699. The soluble gp160 molecules exhibited a binding ability for CD4 on the surface of SupT1 cells that was 33-36% that of the soluble gp120 glycoproteins. These results implicate residues 687-689 and 697-699 as important components of the stop-transfer signal that anchors the gp160 envelope glycoprotein precursor in the membrane. The data also suggest that characteristics in addition to hydrophobicity are required for stop-transfer signals.

Amino Acid Sequence↗

PubMed1989

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.

Antibody-Dependent Cell Cytotoxicity↗

PubMed2000

Interactions between HIV-1 gp41 core and detergents and their implications for membrane fusion.

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.

Amino Acid Sequence↗

PubMed1990

Identification of the most significant amphipathic helix with application to HIV and MHV envelope proteins.

Amphipathic helices, which play important roles in protein structure, occur in a wide variety of lengths. Yet existing methods employ fixed window lengths. We present a hierarchical procedure that identifies the Q most significant amphipathic helices regardless of length. Since the observed hydrophobicities are not normally distributed, test statistics usually employed for least-squares regression are inappropriate for assessing statistical significance of amphipathic helices. We show that an adjusted F statistic provides a good test. An application to the envelope protein of HIV finds an unexpected long amphipathic helix in gp41.

Algorithms↗

PubMed1992

CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins.

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.

Animals↗

PubMed2001

Immunization of mice with recombinant gp41 in a systemic prime/mucosal boost protocol induces HIV-1-specific serum IgG and secretory IgA antibodies.

We tested the immunogenicity in mice of a recombinant fusion protein (gp41HA) consisting of the ectodomain of the HIV-1(IIIB) envelope glycoprotein gp41 fused to a fragment of the influenza virus HA2 hemagglutinin protein. An intraperitoneal prime followed by intranasal or intragastric boosts with gp41HA induced high concentrations of serum IgG antibodies and fecal IgA antibodies that reacted with gp41 in HIV-1(IIIB) viral lysate and were cross-reactive with gp41 in HIV-1(MN) lysate. By indirect immunofluorescence, serum IgG and fecal IgA from immunized mice were also shown to recognize gp41 in acetone-fixed human peripheral blood mononuclear cells infected with either syncytium-inducing (SI) or non-syncytium-inducing (NSI) North American HIV-1 field isolates, but not uninfected cells. Thus, this recombinant antigen may be useful in prime/boost immunization protocols designed to induce systemic and mucosal antibodies that recognize multiple primary HIV-1 isolates.

AIDS Vaccines↗

PubMed2006

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission.

BACKGROUND: HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission. RESULTS: The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs. CONCLUSION: The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.

Adult↗

PubMed1999

T20/DP178, an ectodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte N-formyl peptide receptor.

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein-coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

Cells, Cultured↗

PubMed1988

Comparative analysis of gp41 antigens by enzyme-linked immunosorbent assays for detecting antibodies to human immunodeficiency virus type 1.

We have compared the antigenic qualities of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein with a synthetic oligopeptide (peptide R21S) and a bacterially synthesized protein (protein 566), which are homologous with the N-terminal region of gp41, in enzyme-linked immunosorbent assays (ELISA) for detecting antibodies to HIV-1 in sera of patients with the acquired immunodeficiency syndrome (AIDS) or the aids-related complex (ARC). Although the use of all three types of antigens readily allowed the detection of antibodies in human sera, ELISA employing purified gp41 glycoprotein and the protein 566 were more specific and sensitive than the peptide R21S ELISA.

AIDS-Related Complex↗

PubMedDate not supplied

A trans-dominant mutation in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 inhibits membrane fusion when expressed in target cells.

A recombinant vaccinia virus was used to express a mutation in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120-gp41. In this mutant protein, the second amino acid in the N-terminal region of gp41 has been converted from a hydrophobic valine residue to the polar glutamate. When recombinant vaccinia viruses encoding wild-type HIV-1 envelope glycoprotein infect a lymphocyte cell line lacking CD4, the cells express the HIV-1 envelope glycoprotein gp120-gp41 and are able to fuse with a CD4(4) T lymphocyte cell line. Cells expressing the mutant envelope glycoprotein are unable to fuse with CD4(4) T lymphocytes. When both viruses infect CD4- cells simultaneously, there is an inhibition of fusion to CD4+ cells with an increasing fraction of the virus encoding the mutated envelope glycoprotein. Interestingly, when the opposing, or CD4+ target cells are infected with the mutation-expressing virus, while CD4- cells are infected with wild-type envelope-expressing virus, a similar inhibition of fusion is observed. This suggests that the mutated envelope glycoprotein does not need to reside in the same membrane as the wild-type protein it inhibits.

Antibodies, Monoclonal↗

PubMed1999

Inhibition of human immunodeficiency virus type 1 infectivity by the gp41 core: role of a conserved hydrophobic cavity in membrane fusion.

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an alpha-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.

Amino Acid Sequence↗