Pregnancy and human herpesvirus 8 reactivation in human immunodeficiency virus type 1-infected women

To investigate the impact of pregnancy on human herpesvirus 8 (HHV-8) reactivation in human immunodeficiency virus type 1 (HIV-1)-infected women, the HHV-8 DNA presence and load were analyzed in peripheral blood mononuclear cells (PBMCs) and cervicovaginal secretions (CVSs) from 15 pregnant women coinfected with HIV-1 and HHV-8. HHV-8 detection was analyzed in relation to anti-HHV-8 antibodies and HIV-1-related parameters. Nucleotide sequence analysis of an ORFK1 hypervariable region of the HHV-8 strains was performed. HHV-8 was detected in maternal PBMCs (5/15 women) from the second trimester and in CVSs (5/15 women) mainly from the third trimester. The HHV-8 load significantly increased late in pregnancy in both maternal compartments and was associated with a significant increase in HIV-1 shedding in the genital tract. Antilytic antibodies were significantly more common in HHV-8 DNA-positive women. An elevated HHV-8 load was found in the PBMCs of an infant born to a mother with large amounts of HHV-8 in both compartments at delivery. Different ORFK1 subtypes were found in maternal samples, whereas the same subtype was identified in the mother-child pair. These data suggest that pregnancy may induce HHV-8 replication in HIV-1-infected women. An augmented HHV-8 load may, in turn, influence mother-to-child transmission, since one of the HIV-1-infected mothers with HHV-8 reactivation transmitted her ORFK1 subtype to the infant, who showed a high level of HHV-8 viremia indicative of a primary infection. This finding documents for the first time the perinatal transmission of a specific HHV-8 subtype. Vertical transmission may thus play a role in HHV-8 spread also in areas of subendemicity among HIV-1-infected women.     

抗人免疫缺陷病毒1型gp120人源单链抗体的表达

目的研究人源抗人免疫缺陷病毒１型（ＨＩＶ－１）ｇｐ１２０单链抗体（ＳｃＦｖ）。方法以人工合成的ＨＩＶ－ｌｇｐ１２０Ｖ３环多肽为抗原，利用噬菌体抗体库技术，筛选含有抗－ｇｐ１２０ＳｃＦｖ基因的噬菌体，提取质粒，转化大肠杆菌ＨＢ２１５１，表达可溶性ＳｃＦｖ。结果经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析，表达产物分子量为２８ｋＤ左右，且具有ｃ－ｍｙｃ活性；ＥＬＩＳＡ和Ｄｏｔｂｌｏｔ结果表明，可溶性ＳｃＦｖ具有较好的抗原结合活性和较强的特异性；竞争性ＥＬＩＳＡ实验结果进一步证明表达产物的特异抗－ｇｐ１２０活性。结论该技术便捷有效，可大量获得人源抗ＨＩＶ抗体片段，为进一步研究抗ＨＩＶ抗体的生物活性和ＨＩＶ感染诊治打下基础     

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.     

人免疫缺陷病毒Ⅰ型包膜糖蛋白gp120基因在大肠杆菌中的高效表达

目的提高人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）包膜糖蛋白ｇｐ１２０基因在原核系统中的表达量。方法采用聚合酶链反应（ＰＣＲ）技术扩增出５６０ｂｐ的ＨＩＶ－１ＬＡＶ株ｇｐ１２０Ｎ端基因片段，经ＥｃｏＲⅠ及ＳａｌⅠ酶切后插入高效表达载体ｐＥＴ２８ａ，得到重组质粒ｐＥＴ１２０，并转化表达宿主菌ＢＬ２１（ＤＥ３），经诱导高效表达出ＨＩＶ－１ｇｐ１２０基因片段。结果间接酶联免疫吸附试验（ＥＬＩＳＡ）及Ｗｅｓｔｅｒｎｂｌｏｔ实验表明，表达产物具有良好的抗原性及特异性。ＳＤＳ－ＰＡＧＥ电泳分析结果表明，ｇｐ１２０表达量占总菌体蛋白的５０％。结论在原核系统中高效表达了ＨＩＶ－１ｇｐ１２０基因     

Human immunodeficiency virus gp120 downregulates CD1d cell surface expression

CD1d is an MHC class I-like surface molecule that presents endogenous glycoplipid antigens. The effect of HIV infection on CD1d surface expression has not yet been reported. FACS analysis revealed significantly lower levels of CD1d on CD14(+) monocytes from HIV-infected subjects compared to HIV-infected subjects on HAART and healthy controls. CD1d expression correlated inversely with viral load in infected individuals. CD1d surface expression on human cell lines was downregulated after infection with M-tropic HIV, T-tropic HIV, or after exposure to HIV gp120 in vitro. These data suggest that CD1d-mediated responses are altered during HIV infection and may thus contribute to the global immunodeficiency seen in these patients.     

人免疫缺陷病毒Ⅰ型包膜糖蛋白gp120基因在大肠杆？…

目的 提高人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）包膜糖蛋白ｇｐ１２０基因在原核中的表达量。方法 采用聚合酶链反应（ＰＣＲ）技术扩增出５６０ｂｐ的ＨＩＶ－１ＬＡＶ株ｇｐ１２０Ｎ端基因片段，经ＥｃｏＲⅠ及ＳａｌⅠ酶切后插入高效表达载体ＰＥＴ２８ａ得到重组质粒ｐＥＴ／１２０，并转化表达宿主菌ＢＬ２１（ＤＥ３），经诱导高效表达出ＨＩＶ－１ｇｐ１２０基因片段。结果 间接酶联免疫吸附试验（ＥＬＩＳＡ）及Ｗｅｓｔｅｒ     

Induction of human immunodeficiency virus 1 replication by human herpesvirus 8

BACKGROUND: Human immunodeficiency virus 1 (HIV-1)-infected individuals are commonly infected with herpesviruses, including cytomegalovirus, herpes simplex virus, varicella-zoster virus, and human herpesvirus 8 (HHV-8, also known as Kaposi sarcoma-associated herpesvirus [KSHV]). Previous studies have demonstrated that coinfection with herpesviruses can modulate HIV-1 replication. This can occur either through direct interaction between the 2 viruses or through secondary effects resulting from the release of cellular factors in response to infection. OBJECTIVE: To investigate HIV-1 replication in the presence and absence of HHV-8. DESIGN AND METHODS: HIV-1 replication was analyzed following culture of HIV-1-infected CD4(+) T cells in the presence of HHV-8 infected B-cell lines or control, uninfected B-cell lines. To confirm and extend the results of these in vitro studies, HIV-1-infected T cells were injected into human skin transplanted onto severe combined immunodeficient mice. The human skin was also injected with purified HHV-8 or phosphate-buffered saline as a control and HIV replication measured in biopsy specimens taken 5 to 8 days later. RESULTS AND CONCLUSIONS: The results demonstrated a significant increase in HIV-1 replication in the presence of HHV-8 in both the in vitro and in vivo model systems. Although the mechanism responsible for HHV-8 induction of HIV-1 replication remains to be identified, the results indicate that these 2 viruses may interact at the molecular level in coinfected patients, resulting in increased HIV-1 viral load.     

Different proliferative response of human and chimpanzee lymphocytes after contact with human immunodeficiency virus type 1 gp120

T cell functional defects are a common aspect of human immunodeficiency virus (HIV) infection. Moreover, it has been suggested that indirect mechanisms are involved in CD4⁺ cell depletion. Unresponsiveness to proliferative stimuli of lymphocytes incubated with HIV particles or with viral proteins is well documented. Nevertheless, drawing a clear picture of the anergy phenomenon is difficult because of several unresolved and controversial questions. Here we report that recombinant gp120 induces anergy in T helper lymphocytes cultured with different stimuli. The proliferative responses to interleukin (IL)-2, IL-4, IL-6, anti-CD2, anti-CD3 and phorbol 12-myristate 13-acetate are inhibited. Moreover, anergic cells show a different distribution in cell cycle phases as compared to control cells, leading us to suggest that the progresion in the cell cycle is hampered and that a pre-mitotic block takes place. Furthermore, since chimpanzees are susceptible to HIV-1 infection without showing immunodeficiency signs, we analyzed the proliferation of chimpanzee lymphocytes without observing anergy in cells preincubated with gp120. Taken together, these results support the hypothesis that anergy plays an important role in HIV infection in vivo.     

Human herpesvirus 6 inhibits human immunodeficiency virus type 1 replication in cell culture.

The SF strain of human herpesvirus 6 (HHV-6SF) isolated from the saliva of a human immunodeficiency virus (HIV)-infected individual was shown to inhibit HIV type 1 (HIV-1) replication in both peripheral blood mononuclear cells and purified CD4+ lymphocytes. This suppression of HIV-1 replication led to decreased cytopathic effects of HIV-1 and prolonged survival of CD4+ cells in culture. Even low levels of HHV-6 added to peripheral blood mononuclear cells showed an inhibitory effect on HIV-1 replication. These results differ from those previously reported showing enhanced HIV-1 production following infection with another strain of HHV-6.     

Seroprevalence of human herpesvirus 8 in human immunodeficiency virus 1-positive and human immunodeficiency virus 1-negative populations in Japan

To determine the seroprevalence of human herpesvirus 8 (HHV8) among human immunodeficiency virus 1 (HIV-1)-positive (HIV-1⁺) and HIV-1-negative (HIV-1⁻) populations in Japan, 276 HIV-1⁺ patients and 1,000 HIV-1⁻ blood donors were enrolled in this study. Antibodies against HHV8 latency-associated nuclear antigen (LANA) were examined through indirect immunofluorescent assay by using a B-cell line that was infected latently with HHV8 (body cavity-based lymphoma 1). An HHV8⁻ and Epstein-Barr virus-negative B-cell line (Ramos) was used as a control. Thirty-two seropositive cases against LANA (anti-LANA⁺) were identified among the 276 HIV-1⁺ patients who were studied. Five cases were foreigners living in Japan. The risk factor of all 27 Japanese cases was unprotected sexual intercourse, and the great majority of these cases (23 in 27; 85%) reported homosexual/bisexual behavior. Anti-LANA⁺ status correlated with the presence of sexually transmitted diseases, such as amoeba and HBV infection, further suggesting male homosexual behavior as the main route of HHV8 transmission in Japan. Only two LANA⁺ cases were identified among 1,000 HIV⁻ blood donors in Japan; thus, seroprevalence of HHV8 identified by LANA was estimated to be 0.2% among HIV-1⁻ populations in this country. J. Med. Virol. 57:159–162, 1999. © 1999 Wiley-Liss, Inc.     

Human immunodeficiency virus type 1 gp120 C5 region mimics the HLA class I α1 peptide-binding domain

Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been suggested as one of the possible mechanisms of induction of an autoimmune response by human immunodeficiency viruses. A monoclonal antibody (M38) was previously shown to bind to both human immunodeficiency virus type 1 (HIV-1) gp120 and β-2 microglobulin-free HLA class I heavy chains encoded by an HLA C allele. Using HLA C recombinant proteins and synthetic peptides, the M38 class I binding site was mapped to a stretch of 44 amino acids of the al domain. The amino acid residues recognized are clustered in two non-contiguous regions at positions 66-69 (KYKR) and 79-82 (RKLR) shared by almost all HLA C alleles. On HIV-1 gp120, M38 binds to two non-contiguous sequences (KYK and KAKR) at positions 490-492 and 505-508 located at the edges of a large hydrophobic region that is apparently involved in binding the transmembrane glycoprotein gp41. The C-terminal gp120 M38-reactive region (KAKR) lies within the immunodominant sequence APTKAKRRVVQREKR, against which the majority of HIV-infected individuals produce antibodies. The results indicate that a functionally important region of HIV-1 gp120 shares similar amino acid sequence motifs with the antigen recognition site of most HLA class I C alleles. The molecular mimicry may be the basis for autoimmune responses in HIV infection.     

Enzyme-linked immunoassay for human immunodeficiency virus type 1 envelope glycoprotein 120.

An enzyme-linked immunosorbent assay (ELISA) that can measure picogram quantities of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) in cell culture medium or body fluids has been developed. Recombinant, soluble CD4 immobilized in microtiter trays was used to capture gp120, which was then detected with polyclonal sheep antibody to gp120 followed by biotinylated rabbit anti-sheep immunoglobulin G and an avidin-alkaline phosphatase indicator system. With a reference recombinant gp120, the assay showed a linear relationship between optical density and concentrations ranging from 60 to 6,000 pg/100-microliters well; precision of the assay varied with the concentrations and ranged from +/- 40% with amounts smaller than 200 pg to +/- 10% with amounts larger than 200 pg. In a group of coded samples containing 60 pg (approximately 10(7) molecules) of reference gp120, the assay correctly identified the samples as containing gp120 99% of the time, with no false-positive results recorded for blank samples. Recombinant gp120 prepared in another cell culture system demonstrated a binding coefficient 13-fold lower than that of reference gp120. Mixing standard amounts of reference gp120 with increasing concentrations of human sera reduced assay sensitivity, although the linear relationship between gp120 concentration and optical density remained. With this assay we were able to detect gp120 in HIV-1 suspensions prepared from cultured lymphoblastoid cells and in the sera of HIV-1-infected patients. This ELISA for gp120 should be useful for studying the biological role of gp120 in HIV infection.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

Summary We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wildtype virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.     

Cell surface display of human immunodeficiency virus type 1 gp120 on Escherichia coli by using ice nucleation protein.

A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface of Escherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.     

Protease-defective, gp120-containing human immunodeficiency virus type 1 particles induce apoptosis more efficiently than does wild-type virus or recombinant gp120 protein in healthy donor-derived peripheral blood T cells.

Apoptosis and syncytium formation are two mechanisms by which human immunodeficiency virus type 1 (HIV-1) impairs uninfected CD4+ T-cell function and are mainly involved in the progression of the disease to AIDS. Previously, we showed that gp120-containing, protease-deficient HIV-1 (L-2) particles generated syncytia by particle-mediated fusion with uninfected cultured CD4+ T cells. Here, we present evidence that such L-2 particles can induce apoptosis in 40 to 50% of T cells which were enriched from HIV-1-negative healthy donor-derived peripheral blood mononuclear cells (PBMC-Ts). Activation of PBMC-Ts with phytohemagglutinin, concanavalin A, or ionomycin after incubation with L-2 particles resulted in the loss of proliferative capacity and gradual induction of apoptosis over 3 days. Wild-type strain LAI particles or recombinant gp120 were markedly less efficient (< or = 15%) at inducing such apoptosis. Western blot (immunoblot) analysis revealed that L-2 particles contained a larger amount of Env gp120 than LAI particles. Either preincubation of PBMC-Ts with a Fas antagonist or preincubation of L-2 particles with soluble CD4 blocked most of the apoptosis. This suggests that L-2-like particles can play a major role in HIV-1-induced apoptosis of uninfected bystander cells.     

Human immunodeficiency virus type 1 tropism for human macrophages.

Human immunodeficiency virus (HIV) infects cells of the monocyte/macrophage lineage in addition to lymphocytes, and infection of these cells may be responsible for viral persistence and dissemination, encephalopathy of the acquired immunodeficiency syndrome and other sequelae of HIV infection. We have developed an in vitro model utilizing peripheral-blood monocyte-derived macrophages to study HIV-1 infection of macrophages. HIV-1 isolates vary greatly in their ability to infect and replicate in macrophages, from highly restricted to highly productive infection. Productively infected macrophages undergo syncytium formation but remain viable in culture and support sustained levels of virus production for prolonged periods. Transformed monocytoid and lymphoid cell lines, however, show very different patterns of permissiveness for HIV-1 strains and do not reflect their corresponding primary cell types in studies of host cell tropism. Studies on viral entry show that the CD4 molecule, known to be the HIV receptor on lymphoid cells, is expressed at low levels on the surface of macrophages as well, where it functions as the receptor for viral entry. Therefore, differential host cell tropism does not result from the use of an alternative macrophage-specific receptor instead of CD4.     

Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation

Summary.  Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via α(1–3) or α(1–4) linkages in N-linked glycans of gp 120. [³H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [³H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [³H]-focuse label was also released by treatment of the labelled gp 120 with an α-L-fucosidase specifically removing fucose in α(1–3) and α(1–4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. β(1–4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific α-fucosidase as well as an enzyme specific for α(1–3)- or α(1–4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation. Accepted July 17, 1997 Received December 3, 1996     

Human immunodeficiency virus-1 gp120 and gp160 envelope proteins modulate mesangial cell gelatinolytic activity.

Patients with human immunodeficiency virus (HIV) infection often develop glomerular lesions (mesangial expansion and sclerosis). Modulation of matrix degradation may be important in the expansion of the mesangium. We studied the effect of HIV sera and HIV-1 envelope glycoproteins on gelatinolytic activity of human mesangial cells. HIV serum-treated cells showed lower (P < 0.01) gelatinolytic activity when compared with cells treated with control serum (control serum, 4.3 +/- 0.1 versus HIV serum, 3.3 +/- 0.1 micrograms gelatin degraded/mg protein). Mesangial cells incubated with HIV-1 gp120 protein also showed decreased (P < 0.01) gelatinolytic activity (control, 4.6 +/- 0.2 versus HIV-1 gp120 protein, 1.7 +/- 0.2 micrograms gelatin degraded/mg protein). HIV-1 gp160 protein also inhibited (P < 0.05) mesangial cell gelatinolytic activity as judged by a biotin-avidin assay as well as by a 3H gelatin degradation assay. In contrast, gp alpha-1 acid, a nonviral glycoprotein, did not modulate mesangial cell gelatinolytic activity. These results suggest that the serum contents of HIV patients decrease gelatinolytic activity of mesangial cells. This effect of HIV sera seems to be mediated through HIV-1 gp proteins.     

Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro.

Monoclonal antibodies (MAbs) to a recombinant form of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1 IIIB) were raised in rats and screened for their ability to block recombinant gp120 binding to recombinant, soluble CD4 (sCD4) in vitro. Four such MAbs were identified and characterised. Each MAb bound strongly to gp120 from eight widely divergent HIV-1 strains from the United States and Africa. Two MAbs were mapped to the fourth conserved (C4) region of gp120, whereas the other two recognised an as yet undefined, conformationally sensitive epitope. MAbs to the latter epitope were the more potent in blocking the gp120-sCD4 interaction. None of the MAbs, however, had potent neutralising activity.     

Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.