Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide. HIV Variant Working Group

BACKGROUND: Although numerous mutations that confer resistance to protease inhibitors (PRI) have been mapped for HIV-1 subtype B, little is known about such substitutions for the non-B viruses, which globally cause the most infections. OBJECTIVES: To determine the prevalence of PRI-associated mutations in PRI-naive individuals worldwide. DESIGN: Using the polymerase chain reaction, protease sequences were amplified from 301 individuals infected with HIV-1 subtypes A (79), B (95), B' (19), C (12), D (26), A/E (23), F (26), A/G (11), and H (3) and unclassifiable HIV-1 (7). Amplified DNA was directly sequenced and translated to amino acids to analyze PRI-associated major and accessory mutations. RESULTS: Of the 301 sequences, 85% contained at least one codon change giving substitution at 10, 20, 30, 36, 46, 63, 71, 77, or 82 associated with PRI resistance; the frequency of these substitutions was higher among non-B (91%) than B (75%) viruses (P < 0.0005). Of these, 25% carried dual and triple substitutions. Two major drug resistance-conferring mutations, either 20M or 30N, were identified in only three specimens, whereas drug resistance accessory mutations were found in 252 isolates. These mutations gave distinct prevalence patterns for subtype B, 63P (62%) > 77I (19%) > 10I/V/R (6%) = 361 (6%) = 71T/V (6%) > 20R (2%), and non-B strains, 36I (83%) > 63P (17%) > 10I/V/R (13%) > 20R (10%) > 77I (2%), which differed statistically at positions 20, 36, 63, 71, and 77. CONCLUSIONS: The high prevalence of PRI-associated substitutions represent natural polymorphisms occurring in PRI-naive patients infected with HIV-1 strains of subtypes A-H. The significance of distinct mutation patterns identified for subtype B and non-B strains warrants further clinical evaluation. A global HIV-1 protease database is fundamental for the investigation of novel PRI.     

MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1

Optimal infectivity of HIV-1 virions requires synthesis of the HIV-1 regulatory protein Nef in some producer cells but not others. A survey of 18 lymphoid cell lines found that Nef was dispensable in three, each of which harbored gammaretroviruses. Nef-dependent cell lines were rendered Nef-independent by a cell-free supernatant from the independent lines or by transfection of cloned murine leukemia virus (MLV). Analysis of MLV deletion mutations identified glycosylated gag (glycogag) as the factor that rescues Nef-defective HIV-1 virions. Glycogag was also demonstrated to be required for the infectivity of MLV virions produced in lymphoid cells. Direct comparison of Nef and glycogag revealed identical dependence for activity on Env-pseudotype and producer cell type. The two proteins colocalize within cells, and both increase the yield of viral cDNA in target cells. The functional similarity of Nef and glycogag is a compelling example of convergent evolution in which two structurally unrelated proteins provide a function necessary for virion infectivity in lymphoid cells.Nef is a myristoylated protein encoded by HIV-1, HIV-2, and SIV, crucial for virus replication in vivo and rapid AIDS progression (1–3). It performs a remarkable array of activities by exploiting many of its surfaces to interact with several cellular molecules. By interacting with proteins implicated in intracellular trafficking, it modulates cell surface expression of numerous molecules, including the receptor CD4 (4, 5) and MHC-I (6). Alleles derived from most SIV isolates also down-regulate the TCR/CD3 complex (7–9). In addition, Nef alters the activation threshold of lymphocytes (10–12) by interacting with protein kinases (13–16) and modulates apoptotic signals (10, 17).Nef also has a positive effect on the infectivity of virions (18, 19), a function which remains mechanistically unexplained. Although the ability to down-regulate CD4 can contribute to the effect of Nef on infectivity (20), this activity is visible by using CD4-negative producer cells and was shown to be independent from other Nef effects (18, 21–23), it requires its expression in virus-producing cells and is manifested at an early step of the infection process of target cells (18, 22, 24–26). Nef might play a crucial role during penetration of retroviral cores into the cytoplasm (27). Accordingly, HIV-1 pseudotyped by vesicular stomatitis virus G (VSV-G), which relies on endosomal uptake and, therefore, might enhance cytoplasmic delivery, does not require Nef (28, 29). Although found in virus particles, recent data indicate that Nef itself might not function as a virion protein (30, 31), suggesting that it could induce a yet unknown modification of the particle. The effect of Nef on infectivity was shown to depend on dynamin 2 and clathrin activities in producer cells (32) and on a di-leucine motif critical for the interaction with the clathrin adaptor complexes AP2 (33, 34). The biogenesis and/or trafficking of intracellular vesicles in virus producing cells might therefore play a crucial role in modulating virion infectivity.Most gammaretroviruses encode an accessory protein (gPr80 or glycogag) from unspliced RNA via an alternative CUG initiation codon upstream and in-frame with the standard Gag polyprotein (35–39). As a result, a leader sequence [88 amino acids in MLV provirus genome (MoMLV)] is added to the N terminus of conventional Gag. The protein encoded has a type II transmembrane topology, with the conventional Gag residues being extracellular or located in the lumen of the ER, and glycosylated. Mature gPr80 is proteolytically cleaved, and only half of the conventional Gag sequence remains attached to the integral transmembrane protein (40). Although not strictly required for virus replication in vitro, gPr80 is crucial in vivo for sustained virus replication and disease progression (41–47). The mechanisms engaged by gPr80 to promote virus replication remain largely unknown. However, a recent report has revealed that gPr80 affects release of both MLV and HIV-1 by facilitating budding from lipid rafts (48).In this study, a screen of 18 producer cell lines identified three lymphoid cell lines capable of generating HIV-1 that does not require Nef for maximal infectivity. Glycosylated gag expressed from gammaretrovirus genomes harbored by these cell lines was found to functionally replace the infectivity function of Nef, unveiling a role for glycogag as an infectivity factor.     

B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.     

Significance of HIV-1 genetic subtypes

HIV is the most significant new pathogen that emerged during the twentieth century. Since the recognition of AIDS in 1981, HIV has caused a worldwide epidemic. HIV-1 mutates extensively and shows high genetic diversity and thereby poses significant challenges for effective surveillance and disease control. At the beginning of the 1990s phylogenetic analyses of HIV-1 sequences from different sources of the world epidemic revealed that HIV-1 can be divided into different clades or subtypes. However, most of the knowledge from that time was based on information from western countries, where subtype B predominated. Important questions were raised about the possibility that genetic and phenotypic differences in HIV-1 may affect transmissibility, infectivity and pathogenicity, in addition to responses to therapy and vaccines. On this basis this study was initiated in 1994, and presented as a thesis in 1999. This paper gives an overview of the results from this thesis (based on 6 original papers) and the conclusions drawn. In summary, determination of the genetic subtype of HIV-1 probably has little value for routine clinical care of individual patients, but provides a powerful tool for monitoring changes in local and global transmission patterns.     

HIV-1 MN Env 15-mer peptides better detect HIV-1 specific CD8 T cell responses compared with consensus subtypes B and M group 15-mer peptides

OBJECTIVE: To compare the ability of three Env (15-mer) peptide sets derived from the HIV-1 MN, the subtype B consensus, and the group M consensus to detect HIV-1 specific interferon (IFN)-gamma responses in HIV-1 subtype B infected subjects. METHODS: Peripheral blood mononuclear cells were obtained from 17 HIV-1 subtype B seropositive and 5 HIV-1 seronegative subjects. Peptide matrices comprising each peptide set were used in IFN-gamma Elispot assays to screen for T cell epitopes. Following matrix deconvolution, individual peptides were analyzed by IFN-gamma intracellular cytokine-staining to confirm and characterize the responding cells. RESULTS: HIV specific IFN-gamma responses were detected in 17 of 17 HIV-1 seropositive and none of 5 HIV-1 seronegative subjects by Elispot. Within the 17 HIV-1 seropositives, 16, 14, and 11 subjects responded to MN, B consensus, and group M env peptides, respectively. Responses were confirmed by intracellular cytokine analysis in 14 subjects and were in the CD3CD8 compartment. Cross-recognition of 'equivalent' peptides (i.e., peptides mapping to the same sequence region from the three peptide sets) was observed in 9 of 17 subjects. Peptide set specific responses to individual peptides were also observed; 11, 1, and 1 subjects demonstrated peptide set specific responses to MN, B consensus, and consensus group M, respectively. CONCLUSION: MN derived Env peptides were better able to detect HIV-1 specific CD8 T cell responses, many of which were not detectable by the equivalent clade or group consensus peptides. No single peptide set detected all the IFN-gamma responses within an individual. These results demonstrate the importance of reagent selection for monitoring of HIV responses in HIV-1 infected individuals and subsequently vaccine recipients.     

MSM人群急性HIV-1感染者病毒亚型分析

目的研究2007-2009年男男性行为人群（MSM）艾滋病病毒-1型（HIV-1）的基因亚型,以监测MSM人群HIV病毒株的最新流行情况。方法采集95例MSM急性HIV-1感染者的外周全血,从外周全血浓缩白细胞中提取脱氧核糖核酸（DNA）,用巢式聚合酶链反应（Nested-PCR）对HIV-1的前病毒DNA进行扩增,然后检测并分析PCR扩增产物的核苷酸序列,确定病毒的基因亚型。结果 95例感染者平均年龄为（35.02±9.15）岁,其中20～50岁的青壮年45例（94.74%）,未婚51例（53.68%）,大专或大学及以上文化程度者30例（占31.58%）。感染者中共发现CRF01_AE亚型、B亚型、BC重组亚型（包括CRF07_BC和CRF08_BC）及C亚型4种亚型。其中CRF01_AE重组亚型56例（占58.95%）,B亚型28例（占29.47%）,BC重组亚型10例（占10.53%）,C亚型1例（1.05%）。结论 MSM人群急性HIV-1感染者年轻化,文化程度普遍偏低。小样本流行病学调查显示,急性HIV-1感染者中有CRF01_AE亚型、B亚型、BC重组亚型及C亚型4种亚型病毒株流行,CRF01_AE亚型为MSM人群的主要亚型。     

Transepithelial transport of HIV-1 by M cells is receptor-mediated

Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.     

Inhibition of HIV-1 group M and O isolates by fusion inhibitors

We examined the susceptibility of HIV-1 group M and O isolates to the fusion inhibitors T-20 and T-1249. Unexpectedly, HIV-1 O isolates were as sensitive as group M viruses to inhibition by T-20 but were usually less susceptible to T-1249. Our data suggest that T-20 has broad antiretroviral activity and would be effective in individuals with HIV-1 O infection. However, polymorphisms in gp41 might affect the sensitivity of HIV-1 O to second-generation fusion inhibitors.     

Catalytic efficiency and vitality of HIV-1 proteases from African viral subtypes

The vast majority of HIV-1 infections in Africa are caused by the A and C viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. Because some amino acid polymorphisms occur at sites that have been associated with drug resistance in the B subtype, it is important to assess the effectiveness of protease inhibitors that have been developed against different subtypes. Here we report the enzymatic characterization of HIV-1 proteases with sequences found in drug-naive Ugandan adults. The A protease used in these studies differs in seven positions (I13V/E35D/M36I/R41K/R57K/H69K/L89M) in relation to the consensus B subtype protease. Another protease containing a subset of these amino acid polymorphisms (M36I/R41K/H69K/L89M), which are found in subtype C and other HIV subtypes, also was studied. Both proteases were found to have similar catalytic constants, k(cat), as the B subtype. The C subtype protease displayed lower K(m) values against two different substrates resulting in a higher (2.4-fold) catalytic efficiency than the B subtype protease. Indinavir, ritonavir, saquinavir, and nelfinavir inhibit the A and C subtype proteases with 2.5-7-fold and 2-4.5-fold weaker K(i)s than the B subtype. When all factors are taken into consideration it is found that the C subtype protease has the highest vitality (4-11 higher than the B subtype) whereas the A subtype protease exhibits values ranging between 1.5 and 5. These results point to a higher biochemical fitness of the A and C proteases in the presence of existing inhibitors.     

The epidemiology of HIV-1 subtypes in infected patients from northeastern Thailand

In total, 117 HIV-1 infected patients from several provinces in Northeastern Thailand were analysed. All blood samples collected from individuals were confirmed by EIA and Western blot and partially by HIV-1 gag-, pol- and env-PCR. By serotyping with a V3-peptide ELISA, 108 (92.3%) of the sera samples belonged to subtype E, 9 (7.7%) were serotype B. For 10 Thai HIV-1 infections, the serotype and genotype were determined. The genotype was determined by phylogenetic analysis of directly sequenced PCR amplicons, 8 were subtype E, 2 subtype B. For these patients the serotype did correlate with the genotype. Tracing back the origin of Thai patients, it seems that most were infected within early years of the epidemic and the Thai subtype B infected patients have been imported directly from foreign countries via sexual contact. The findings suggest there are two district subtypes in Thailand with the majority being subtype E. The relatively high prevalence of subtype B in Northeastern Thailand may be due to the increasing intermix of the two strains (subtypes E and B) and the migration for employment from foreign countries. This may lead to public health concerns regarding surveillance of HIV-1 subtypes and the regulation of potentially infected workers returning from abroad to the country.     

HIV-1 subtypes in pregnant women in the UK

We analyse the distribution of HIV-1 subtypes in HIV-1-seropositive samples from 333,270 residual neonatal dried blood spot samples tested for routine newborn screening tests in the UK between July 1999 and December 2002. Of the 813 antibody-positive samples shown to contain passively acquired, maternal HIV-1 for which subtyping was attempted, 333 (41%) could not be subtyped due to cross-reactivity or low values of the assay results, and 480 (59%) were classified as B (35, 7.3%) or non-B (445, 92.7%). The proportions of subtyped B samples differed significantly (P=0.004) between those from neonates whose mothers were born in the UK (21.4%) and those from neonates whose mothers were known to be born abroad (7%). Using a serological approach to establish viral serotype, we document the distribution of HIV-1 subtypes in infected pregnant women in the UK.     

河南省人免疫缺陷病毒流行株的基因亚型分析

目的　研究流行于河南省献血员中的人免疫缺陷病毒（ＨＩＶ） 外膜蛋白（ｅｎｖ） 基因亚型。方法　提取ＨＩＶ 感染者外周血单个核白细胞（ＰＢＭＣ） 的ＤＮＡ,经套式聚合酶链反应（ＮｅｓｔｅｄＰＣＲ） 扩增ｅｎｖ 基因片断,对Ｃ２Ｖ３ 区３５０ ～４５０ 核苷酸顺序进行测定和分析。结果　２０ 份样品的ｅｎｖ 基因序列与Ｂ亚型最为接近,基因离散率为８ ．３４ ％ ,与其它国际亚型的基因离散率在２０ ％ 以上;与流行于云南的Ｂ 亚型毒株基因离散率为４ ．１８ ％ ;样品间的基因离散率为３ ．４ ％ 。结论　流行于河南省献血员中的ＨＩＶ 属Ｂ亚型,并与云南的流行株密切相关;其在河南的流行时间在３ ～６ 年间。     

HIV-1 incorporates ABO histo-blood group antigens that sensitize virions to complement-mediated inactivation

ABO histo-blood group antigens have been postulated to modify pathogen spread through the action of natural antibodies and complement. The antigens are generated by a polymorphic glycosyl-transferase encoded by 2 dominant active and a recessive inactive allele. In this study we investigated whether ABO sugars are incorporated into the envelope of HIV-1 virions. HIV vectors derived from cells expressing ABO antigens displayed sensitivity to fresh human serum analogous to ABO incompatibility, and ABO histo-blood group sugars were detected on the viral envelope protein, glycoprotein 120 (gp120). Moreover, lymphocyte-derived virus also displayed serum sensitivity, reflecting the ABO phenotype of the host when cultured in autologous serum due to adsorption of antigens to cell surfaces. Serum sensitivity required both active complement and specific anti-ABO antibodies. Thus, incorporation of ABO antigens by HIV-1 may affect transmission of virus between individuals of discordant blood groups by interaction with host natural antibody and complement.     

A hematopoietic cell L-selectin ligand that is distinct from PSGL-1 and displays N-glycan-dependent binding activity

Human hematopoietic progenitor cells express L-selectin and also express PSGL-1, a ligand for all selectins. Using a shear-based adhesion assay, a hematopoietic cell L-selectin ligand (HCLL) that is expressed on the hematopoietic cell line KG1a and on normal human hematopoietic progenitors was previously identified. To characterize the structural biology of HCLL and to define its relationship to PSGL-1, the effects of chemical and enzymatic treatments on HCLL activity of KG1a cells and membrane preparations were analyzed. Protease digestions and chemical treatments of KG1a cells and membranes indicated that HCLL is an integral membrane glycoprotein. Glycosidase digestions of membrane protein preparations and metabolic treatments of KG1a cells with glycosylation processing modifiers revealed that L-selectin binding determinants on HCLL are sialofucosylated structures presented on complex-type N-glycans. Adhesion assays and biochemical studies showed that this glycoprotein is also expressed on circulating blasts in native acute leukemias. HCLL is distinguishable from PSGL-1: (1) KG1a cells sorted for PSGL-1 expression had equivalent HCLL activity; (2) anti-PSGL-1 blocking antibodies and proteases known to eliminate L-selectin binding to PSGL-1 had no effect on HCLL binding activity of KG1a cells; (3) blasts from native leukemias with low expression of PSGL-1 and CD34 display high HCLL activity; and (4) despite high level expression of PSGL-1, HCLL activity was absent on HL60 cells. These data provide first evidence of a naturally expressed membrane L-selectin ligand expressing binding determinant(s) on an N-linked glycoconjugate. This novel ligand may help mediate L-selectin-dependent cell-cell adhesive interactions within the cytoarchitecture of the bone marrow microenvironment. (Blood. 2000;96:2765-2774)