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The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G 436 WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G 436 WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.

Background Previous studies of the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on HIV-1 replication in macrophages have had inconsistent results, variously reporting no effect, augmentation or inhibition of viral replication. Objective To investigate the regulation of HIV-1 in monocyte-derived macrophages (MDM) by GM-CSF in vitro. Methods The role of GM-CSF on HIV-1 replication was assessed as supernatant and intracellular p24 antigen concentrations and by HIV-1 DNA and mRNA production under different culture conditions. Expression of CD4 and CCR5 receptors was examined. The effect of GM-CSF with an E21R mutation, which binds only to the alpha-chain of GM-CSF receptor, was used as an additional control. Results GM-CSF consistently suppressed HIV-1 replication in human MDM in vitro, as assessed by supernatant and intracellular p24 antigen concentrations and HIV-1 gag mRNA expression. The inhibitory effect of GM-CSF on HIV-1 replication was observed regardless of HIV-1 strain, source of GM-CSF, stage of MDM maturation or timing of GM-CSF exposure in relation to HIV-1 infection. The effect was dose dependent and reversed by addition of a neutralizing monoclonal antibody (4D4). Flow cytometric analysis of surface expression of CD4 and CCR5 indicates that GM-CSF does not affect HIV-1 entry into MDM. Analysis of intracellular HIV-1 DNA and mRNA suggests that HIV-1 replication is inhibited at or before transcription. E21R GM-CSF had no effect on HIV-1 replication in MDM. Conclusions GM-CSF regulates HIV-1 replication in MDM, inhibiting HIV-1 replication through binding to the beta-chain of the GM-CSF receptor.

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.

HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate—labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease. J. Leukoc. Biol. 56: 318–327; 1994.

The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp(666), Trp(672), Phe(673), and Ile(675) in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by approximately 10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu(537) with Trp(666), Trp(672), Phe(673), or Ile(675) abolished entry with 50-80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an approximately 80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.

Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment.

Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41.

Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway.

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.

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.

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.

Peripheral blood monocytes are resistant to productive human immunodeficiency virus type 1 (HIV-1) infection in vitro immediately after isolation. No viral cDNA (either early or late transcripts) was detected by PCR in monocytes exposed to virus on the day of isolation. In contrast, in monocytes cultured for as little as 1 day, initiated and completed reverse transcripts were readily detectable within 24 h of infection with both HIV-1(Ba-L) and primary isolates. The levels of initiated, partially completed, and completed viral DNA copies found 24 h after infection increased progressively with time in culture before infection. Unlike quiescent T lymphocytes, there appeared to be no block or delay in the integration of viral DNA into the genome of susceptible cultured monocytes. With an Alu-PCR method designed to specifically detect proviral DNA being used, integration events were found within 24 h of infection in monocytes cultured for a day or more after isolation. No integration signal was found in freshly isolated monocytes up to 7 days following exposure to the virus. Cloning and sequencing of Alu-PCR-amplified DNA confirmed integration in HIV-1-infected cultured monocytes. Our finding that in vitro replication of HIV-1 is clearly blocked prior to the initiation of reverse transcription in freshly isolated peripheral blood monocytes suggests that these cells may not be susceptible to infection in vivo. Further studies to clarify this possibility and the nature of the block to infection should provide useful information for treatment strategies against HIV-1.

Monocytes from HIV-seronegative persons were analyzed for CD4 expression and susceptibility to infection with HIV-1 on the day of isolation and following 1, 2, and 7 days in culture. Although surface CD4 was readily detected on freshly isolated monocytes, these cells were relatively resistant to infection. After 1 to 2 days in culture, when surface expression of CD4 had decreased over 90% to near background levels, cells became susceptible to infection with HIV-1. CD4 expression on monocytes cultured for 7 days was more than four times higher than that on freshly isolated cells, and the cultured cells were fully permissive to infection. These observations suggest that the differing susceptibility of monocytes and monocyte-derived macrophages to infection with HIV-1 is not simply proportional to the level of surface CD4 expression.

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection. J. Leukoc. Biol. 56: 289–293; 1994.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.