Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H

Most efforts to characterize sequence variation of HIV isolates has been directed toward the structural envelope gene. Few studies have evaluated the sequence variability of auxiliary genes such as nef. In this study 41 new HIV-1 strains, representing the majority of the described envelope subtypes of HIV-1 (A to H), were genetically characterized in the nef region. Phylogenetic analysis showed that 34 strains could be classified in the same subtype in nef and env, and 7 (19%) of the 41 new viruses were recombinants. For two of the seven strains, recombination occurred upstream of the nef gene, whereas for five of the seven strains recombination occurred within the nef gene with a crossover close to the 5' end of the LTR (long terminal repeat). The low intersubtype distance between subtype B and D in the nef gene confirms previous observations in the pol, env, and gag genes, which suggest a common ancestor for these subtypes. The majority of all the previously described functional domains in the nef gene were relatively conserved among the different subtypes, with only minor differences being observed. The myristoylation signal among the different subtypes, with only minor differences being observed. The myristoylation signal was less conserved for subtype C, with one or more amino acid changes being observed at positions 3, 4, and 5. The highly conserved acidic region (positions 62 to 65), critical for the enhancement of viral synthesis with an increased virus growth rate, was less conserved among the subtype G strains from our study. At least three epitopic regions of the nef gene have been defined and each can be recognized by CTLs under a variety of HLA restrictions; all were also relatively well conserved between the different genetic subtypes. Despite the relatively important genetic variation in nef sequences obtained among the different genetic subtypes, functional domains and CTL epitopes were relatively well conserved. In vitro and/or in vivo studies are necessary to study the relevance of the observed differences.     

Genomic diversity in the regulatory nef gene sequences in Indian isolates of HIV type 1: emergence of a distinct subclade and predicted implications

The regulatory functional nef gene is known to mediate a cascade of events during pathogenesis in HIV infection. Variability in the nef gene sequences of HIV-1 A and B subtypes has been well documented. Reasonable data are also available on the pattern of genomic changes in the nef gene of African strains of HIV-1 subtype C, but very little is known about heterogeneity in the nef gene of Indian strains of HIV-1 subtype C, which accounts for 90% of the estimated 5.2 million cases of HIV infection in India. This is a huge number and, therefore, it is important to reveal the extent of sequence variability in the nef gene of HIV-1 subtypes circulating in different parts of India. We carried out full-length nef gene (approximately 620 bp) sequencing on a large number of clinical isolates of HIV-1 circulating in different geographic regions of India. Comparative and phylogenetic analysis revealed 88% (38/43) of cases was HIV-1 subtype C; four cases were diagnosed as subtype A and only one as subtype B. Although most of the crucial functional motifs of the nef gene were conserved, we did observe a few important variations in juxtapositions to functional domains. Interestingly, analyzed nef sequences showed an evolving pattern of segregation away from those reported from other parts of the world, to form a distinct Indian subclade. Deduced amino acid (aa) sequences used to predict HLA binding epitopes for consensus nef gene sequences of Indian strains of HIV-1 revealed two HLA subtype binding domains, GAFDLSFFL (at aa 83) and LTFGWCFKL (at aa 136), in high frequency. The findings from the present study may encourage use of nef gene in molecular diagnostics/genotyping, keeping track of the evolutionary trend and pinpointing the emergence of recombinant strains, and in the future, designing a multiepitope HIV vaccine suitable for the Indian population.     

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.     

Molecular analysis of the full-length genome of HIV type 1 subtype I: evidence of A/G/I recombination.

Phylogenetic analysis of partial env sequences of HIV-1 isolates from Cyprus and Greece suggested the existence of a distinct subtype of the virus, designated as I. We examined whether this subtype represents a distinct group, or a mosaic consisting of previously characterized subtypes. The full-length sequences under consideration were recovered from serum samples of "subtype I" obtained from two nonepidemiologically linked HIV-1-infected subjects in Greece. The first subject was an intravenous drug user (IDU), while the second was a vertically infected child born in 1984 whose parents were both IDUs. A variety of methods, such as diversity plots as well as phylogenetic and informative site analyses, were used to classify the DNA sequences. Subsequent detailed analysis revealed a unique genomic organization composed of alternating portions of subtypes A, G, and I. The two Greek isolates formed a distinct group in most of the pol, gp120, and gp41 regions, and in the vif/vpr, vpu, LTR, and 5' terminus of nef. In contrast, different parts of env and gag as well as the 3' pol region, and the first exons of tat and rev, appeared to have arisen from subtypes A and G. Our results indicate that subtype I, which was probably circulating in Greece in the early 1980s, is a triple mosaic consisting of A, G, and I sequences.     

Full-length genome characterization of an HIV type 2 isolate from India

An infectious Indian human immunodeficiency virus-2 (HIV-2) subtype A isolate was completely sequenced and analyzed and its phylogenetic relatedness was investigated. The unusual limited size of the long terminal repeat (LTR) from the isolate was caused due to a truncation within the nef open reading frame (ORF) located at the U3 region of the LTR. The genetic relatedness and lineage of this HIV-2 strain were investigated. The close relatedness of this isolate to West African HIV-2 isolates confirms a geographical entry route of HIV-2 to this part of the Indian subcontinent. This is the first report of an HIV-2 full genome analysis from the Indian subcontinent as well as from Asia.     

Construction of an infectious HIV type 1 molecular clone from an African patient with a subtype D/C Recombinant Virus

The majority of HIV-1 infections worldwide occur in Africa, where subtype B viruses are rare and intersubtype recombinants are common. Pathogenesis and vaccine studies need to focus on viruses derived from African patients, and infectious HIV-1 molecular clones can be useful tools. To clone non-B subtypes and recombinant viruses from patients, we cultivated HIV-1 from the plasma of a Kenyan long-term survivor. Viral DNA was cloned into a plasmid, which was transfected into COS cells; progeny virus was propagated in PBMCs. Sequence analyses revealed that both the patient's plasma HIV-1 RNA and the cloned DNA genomes were recombinants between subtypes D and C; subtype C sequences comprised the nef and LTR regions. The cloned virus used the CCR5 coreceptor and did not form syncytia in vitro. This infectious HIV-1 subtype D/C recombinant molecular clone obtained from a Kenyan long-term survivor promises to be useful to study pathogenesis and vaccine design.     

Variable sequences in the long terminal repeat and Its downstream region of some of HIV Type 1 CRF01_AE recently distributing among Thai carriers.

Human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences in and downstream of the 5' long terminal repeat (LTR) were compared among samples obtained from 13 HIV-1 CRF01_AE-infected individuals in Thailand from 1998 to 1999. Eleven individuals had highly conserved sequences compared with previously reported CRF01_AE viruses. However, T cell-specific factor (TCF)-1alpha motif, which is located just beside the 3' terminus of the nef sequence, was duplicated in 2 out of the 13 subjects, one of whom had also lost the 24 nucleotides next to the 3' of the primer-binding site. Thus, several characteristics of CRF01_AE LTR and gag-leader sequence were identified in some samples recently obtained in Thailand.     

HIV type 1 subtype C gag and nef diversity in Southern Africa

Several HIV-1 subtype C-specific gag- and/or nef-based vaccines are currently intended for clinical trial in southern Africa. Here we provide sequences of 64 gag and 45 nef genes sampled in Malawi, Zambia, Zimbabwe, and South Africa and assess the degree of southern African HIV-1 diversity that will confront these vaccines. Whereas reasonable phylogenetic evidence exists for geographical clustering of subtype C gag and nef sequences from various other parts of the world, there is little evidence of similar population founder effects in the southern African epidemic. The entire breadth of subtype C diversity is represented in the southern African genes suggesting there may be no advantage in producing region- or country-specific subtype C vaccines. We do not, however, find much evidence of intersubtype recombination in the Southern African genes, implying that the likelihood of vaccine failure due to the emergence of intersubtype recombinants is probably low.     

Monophyletic clade of HIV-1 subtype B in Korea: evolutionary pressure or single introduction?

We have previously shown that many of the nef sequences from Korean HIV-1 subtype B carriers were grouped together in phylogenetic tree analyses. To determine whether this pattern was originated from either a single HIV-1-infected person or some biological pressure which directs the HIV-1 genomic mutation by the Korean specific immunological characters, we analyzed nef sequences from HIV-1-infected individuals with different time intervals. Thirty-two out of 46 analyzed patients formed a Korean monophyletic (KM) clade with 93% bootstrapping value. Eighteen patients' nef sequences were analyzed 1-9 years after first analysis. None of the patients shifted their clade from the first clustered clade (KM or non-KM), and all of the re-analyzed isolates were clustered close to the first analyzed clade. Isolates of the KM dade and non-KM clade in nef analysis showed the same pattern as in env analysis. Phylogenetic clustering evidences from both nef and C2/V3 trees strongly support the idea that introduction of the KM clade in subtype B strains originated from a common source. Thus, treatment and development of an AIDS vaccine may be somewhat easier than in other countries with multiple strains of HIV-1.     

Characterization of a long terminal repeat region from an infectious Indian HIV type 2 isolate

An infectious Indian human immunodeficiency virus type 2 isolate from Mumbai, propagated in this laboratory, was found to bear an unusually short long terminal repeat (LTR) region. Complete sequencing of the 601 bp LTR indicated a loss of around 250 nucleotide pairs from the unique 3' (U3) region as compared to other well-characterized HIV-2 isolates. Phylogenetic analysis of this LTR shows closest relatedness to the Guinea-Bissau subtype A isolates HIV-2(CAM2) and HIV-2(ALI). The LTR from the biologically active infectious clone with the observed deletion contained all functionally relevant promoter and polyadenylation sequences.     

Conserved domains of subtype C nef from South African HIV type 1-infected individuals include cytotoxic T lymphocyte epitope-rich regions.

We have characterized 43 nef sequences from subtype C HIV-1-infected South Africans and compared deduced amino acid sequences with other subtypes to identify areas of conservation. Our Nef amino acid sequences were aligned with a consensus subtype B, HXB2 reference strain and a consensus subtype C sequence. All were found to be highly homologous to subtype B in the central region of Nef, but more variable at the N and C termini of the molecule. Alignment of a consensus amino acid sequence generated from South African subtype C Nef with subtypes A, B, and D underscores cross-clade conservation in the central domain of the molecule. This domain is also rich in previously described cytotoxic T lymphocyte (CTL) epitopes that are restricted by commonly found HLA molecules in the South African population.     

Identification of CRF10_CD viruses among bar and hotel workers in Moshi, Northern Tanzania

We recently identified an HIV-1 subtype C and D circulating recombinant form (CRF10_CD) in infants in Dar es Salaam, Tanzania. So far, this is the only reported HIV-1 CRF in East Africa. However, evidence for its spread in the adult population is scarce. Here we describe the presence of CRF10_CD in two asymptomatic bar and hotel workers in Moshi, Northern Tanzania. Subgenomic sequences from gag (3'p24-5'p7), env (C2-C5), and the 5' LTR were used for phylogenetic analysis and identification of recombination. Genetic divergence between the CRF10_CD sequences from Moshi suggested that they were contracted from independent sources. A third bar worker was infected with an apparent CRF10_CD/subtype A recombinant virus. Our data indicate that CRF10_CD genomes can be transmitted both vertically and heterosexually.     

Polymorphisms in Nef associated with different clinical outcomes in HIV type 1 subtype C-infected children

The human immunodeficiency virus type 1 (HIV-1) negative factor, or Nef, has a variety of functions that are important in viral pathogenesis. Sequence analysis has identified nef mutations that are linked to the rate of disease progression in adults and children infected with HIV-1 subtype B. Here we have sequenced and analyzed HIV-1 subtype C nef sequences from 34 children with rapid (RP) or slow progressing (SP) disease and identified polymorphisms associated with disease stage including motifs involved in specific pathogenic functions. Unlike subtype B, insertions and deletions in the N-terminal variable region were observed exclusively in SP children (8 out of 25). Strong positive selection pressures were found in sites of known functional importance among SP sequences, whereas RP had strong negative selection across the gene. A lineage analysis of selection pressures indicated weaker pressure across the nef gene in SP sequences bearing a deletion in region 8-12, suggesting this deletion has functional importance in vivo. Together these results suggest a differential adaptation of certain Nef functions related to disease progression, some of which may be attributable to immune-imposed pressures. These data broadly reflect previous studies on subtype B, corroborate the decreased cytopathicity of SP viruses, but also highlight potential subtype differences that require further investigation.