Enhanced detection of human immunodeficiency virus type 1 (HIV-1) Nef-specific T cells recognizing multiple variants in early HIV-1 infection

A human immunodeficiency virus (HIV)-preventive vaccine will likely need to induce broad immunity that can recognize antigens expressed within circulating strains. To understand the potentially relevant responses that T-cell based vaccines should elicit, we examined the ability of T cells from early infected persons to recognize a broad spectrum of potential T-cell epitopes (PTE) expressed by the products encoded by the HIV type 1 (HIV-1) nef gene, which is commonly included in candidate vaccines. T cells were evaluated for gamma interferon (IFN-gamma) secretion using two peptide panels: subtype B consensus (CON) peptides and a novel peptide panel providing 70% coverage of PTE in subtype B HIV-1 Nef. Eighteen of 23 subjects' T cells recognized HIV-1 Nef. In one subject, Nef-specific T cells were detected with the PTE but not with the CON peptides. The greatest frequency of responses spanned Nef amino acids 65 to 103 and 113 to 147, with multiple epitope variants being recognized. Detection of both the epitope domain number and the response magnitude was enhanced using the PTE peptides. On average, we detected 2.7 epitope domains with the PTE peptides versus 1.7 domains with the CON peptides (P = 0.0034). The average response magnitude was 2,169 spot-forming cells (SFC)/10(6) peripheral blood mononuclear cells (PBMC) with the PTE peptides versus 1,010 SFC/10(6) PBMC with CON peptides (P = 0.0046). During early HIV-1 infection, Nef-specific T cells capable of recognizing multiple variants are commonly induced, and these responses are readily detected with the PTE peptide panel. Our findings suggest that Nef responses induced by a given vaccine strain before HIV-1 exposure may be sufficiently broad to recognize most variants within subtype B HIV-1.     

Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.

The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.     

中国HIV-1 B’亚型毒株不同基因区与全长基因组系统进化比较

本研究旨在探索不同基因区的使用对HIV-1 B’亚型毒株系统进化分析结果的影响。首先利用既往研究中已发表的共计47条来自泰国,缅甸和中国多个地区不同传播途径的B’毒株近全长基因组序列,将其按基因区分为不同的数据集 (gag, pol, vif, vpr, vpu, env, nef),并分别进行系统进化分析研究。比较不同基因区系统进化分析的结果发现,B’亚型毒株 pol基因在分析的基因区中,具有最低的复杂度和进化速率,可以较好的区分B’TH和B’YN毒株,重复近全长基因组序列的分析结果;尽管env基因则具有最高的复杂度和进化速率,但无法获得类似结果。本研究比较了不同基因区对HIV-1 B’亚型毒株系统进化分析结果的影响,对进一步开展HIV分子流行病学调查,分析我国B’毒株在我国的传播奠定了基础。     

Phylodynamics of HIV-1 Subtype C Epidemic in East Africa

The HIV-1 subtype C accounts for an important fraction of HIV infections in east Africa, but little is known about the genetic characteristics and evolutionary history of this epidemic. Here we reconstruct the origin and spatiotemporal dynamics of the major HIV-1 subtype C clades circulating in east Africa. A large number (n = 1,981) of subtype C pol sequences were retrieved from public databases to explore relationships between strains from the east, southern and central African regions. Maximum-likelihood phylogenetic analysis of those sequences revealed that most (>70%) strains from east Africa segregated in a single regional-specific monophyletic group, here called C_EA. A second major Ethiopian subtype C lineage and a large collection of minor Kenyan and Tanzanian subtype C clades of southern African origin were also detected. A Bayesian coalescent-based method was then used to reconstruct evolutionary parameters and migration pathways of the C_EA African lineage. This analysis indicates that the C_EA clade most probably originated in Burundi around the early 1960s, and later spread to Ethiopia, Kenya, Tanzania and Uganda, giving rise to major country-specific monophyletic sub-clusters between the early 1970s and early 1980s. The results presented here demonstrate that a substantial proportion of subtype C infections in east Africa resulted from dissemination of a single HIV local variant, probably originated in Burundi during the 1960s. Burundi was the most important hub of dissemination of that subtype C clade in east Africa, fueling the origin of new local epidemics in Ethiopia, Kenya, Tanzania and Uganda. Subtype C lineages of southern African origin have also been introduced in east Africa, but seem to have had a much more restricted spread.     

Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication

The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

Candidate Vaccine Sequences to Represent Intra- and Inter-Clade HIV-1 Variation

A likely key factor in the failure of a HIV-1 vaccine based on cytotoxic T lymphocytes (CTL) is the natural immunodominance of epitopes that fall in variable regions of the proteome, which both increases the chance of epitope sequence mismatch with the incoming challenge strain and replicates the pathogenesis of early CTL failure due to epitope escape mutation during natural infection. To identify potential vaccine sequences to focus the CTL response on highly conserved epitopes, the whole proteomes of HIV-1 clades A1, B, C, and D were assessed for Shannon entropy at each amino acid position. Highly conserved regions in Gag (cGag-1, Gag 148–214, and cGag-2, Gag 253–331), Env (cEnv, Env 521–606), and Nef (cNef, Nef 106–148) were identified across clades. Inter- and intra-clade variability of amino acids within the regions tended to overlap, suggesting that polyvalent representation of consensus sequences for the four clades would allow broad HIV-1 strain representation. These four conserved regions were rich in both known and predicted CTL epitopes presented by a breadth of HLA types, and screening of 54 persons with chronic HIV-1 infection revealed that these regions are commonly immunogenic in the context of natural infection. These data suggest that vaccine delivery of a 16-valent mixture of these regions could focus the CTL response against conserved epitopes that are broadly representative of circulating HIV-1 strains.     

Sequence Variations in Human Immunodeficiency Virus Type 1 Nef Are Associated with Different Stages of Disease

nef alleles derived from a large number of individuals infected with human immunodeficiency virus type 1 (HIV-1) were analyzed to investigate the frequency of disrupted nef genes and to elucidate whether specific amino acid substitutions in Nef are associated with different stages of disease. We confirm that deletions or gross abnormalities in nef are rarely present. However, a comparison of Nef consensus sequences derived from 41 long-term nonprogressors and from 50 individuals with progressive HIV-1 infection revealed that specific variations are associated with different stages of infection. Five amino acid variations in Nef (T15, N51, H102, L170, and E182) were more frequently observed among nonprogressors, while nine features (an additional N-terminal PxxP motif, A15, R39, T51, T157, C163, N169, Q170, and M182) were more frequently found in progressors. Strong correlations between the frequency of these variations in Nef and both the CD4(+)-cell count and the viral load were observed. Moreover, analysis of sequential samples obtained from two progressors revealed that several variations in Nef, which were more commonly observed in patients with low CD4(+)-T-cell counts, were detected only during or after progression to immunodeficiency. Our results indicate that sequence variations in Nef are associated with different stages of HIV-1 infection and suggest a link between nef gene function and the immune status of the infected individual.     

CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression

The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.     

Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection.

Studies with the simian immunodeficiency virus have shown that nef deletion results in a low level of viremia and a lack of disease progression in monkeys. Given the similarity of this clinical profile to that observed in long-term survivors of human immunodeficiency virus type 1 (HIV-1) infection, we sought to examine the nef gene in 10 patients who are clinically healthy and immunologically normal despite 12 to 15 years of infection. PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells obtained from long-term survivors. We found that there is no gross deletion within nef in the cases studied; most nef sequences (91.1%) obtained from 10 subjects contained a full-length and intact open reading frame. In addition, at the protein level, there were no discernible differences between the Nef consensus sequences derived from long-term survivors and those from patients with AIDS. We therefore conclude that deletion of or gross sequence abnormality within nef is not likely to be a common explanation for the well-being of long-term survivors of HIV-1 infection. Moreover, phylogenetic analysis of nef sequences suggests that HIV-1 strains found in our study subjects do not have a common origin.     

Human Immunodeficiency Virus Type 1 (HIV-1) Subtype B Epidemic in Panama Is Mainly Driven by Dissemination of Country-Specific Clades

The Human immunodeficiency virus type-1 (HIV-1) subtype B is the most predominant clade in Central America; but information about the evolutionary history of this virus in this geographic region is scarce. In this study, we reconstructed the spatiotemporal and population dynamics of the HIV-1 subtype B epidemic in Panama. A total of 761 HIV-1 subtype B pol sequences obtained in Panama between 2004 and 2013 were combined with subtype B pol sequences from the Americas and Europe. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in Panama derived from the dissemination of multiple founder viruses. Most Panamanian subtype B viruses (94.5%) belong to the pandemic viral strain proposed as originated in the US, whereas others (5.5%) were intermixed among non-pandemic Caribbean strains. The bulk (76.6%) of subtype B sequences from Panama grouped within 12 country-specific clades that were not detected in other Central American countries. Bayesian coalescent-based analyses suggest that most Panamanian clades probably originated between the early 1970s and the early 1980s. The root location of major Panamanian clades was traced to the most densely populated districts of Panama province. Major Panamanian clades appear to have experienced one or two periods of exponential growth of variable duration between the 1970s and the 2000s, with median growth rates from 0.2 to 0.4 year^{− 1}. Thus, the HIV-1 subtype B epidemic in Panama is driven by the expansion of local viral strains that were introduced from the Caribbean and other American countries at an early stage of the AIDS pandemic.     

Broadly increased sensitivity to cytotoxic T lymphocytes resulting from Nef epitope escape mutations

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8(+) CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.     

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.     

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors

It has been reported that patients infected with nef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function of nef alleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derived nef alleles were subcloned into a nef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. All nef alleles from LTNPs showed reduced enhancement in the infectivity of nef-defective HIV-1 mutants compared to the nef alleles of progressors or ACs. Second, we found that nef alleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, all nef alleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1 nef alleles of LTNPs. It awaits further study to conclude that these characteristics of nef alleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.     

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.     

Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the Step study

T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial registration: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.     

HIV-1 nef protein structures associated with brain infection and dementia pathogenesis

The difference between regional rates of HIV-associated dementia (HAD) in patients infected with different subtypes of HIV suggests that genetic determinants exist within HIV that influence the ability of the virus to replicate in the central nervous system (in Uganda, Africa, subtype D HAD rate is 89%, while subtype A HAD rate is 24%). HIV-1 nef is a multifunctional protein with known toxic effects in the brain compartment. The goal of the current study was to identify if specific three-dimensional nef structures may be linked to patients who developed HAD. HIV-1 nef structures were computationally derived for consensus brain and non-brain sequences from a panel of patients infected with subtype B who died due to varied disease pathologies and consensus subtype A and subtype D sequences from Uganda. Site directed mutation analysis identified signatures in brain structures that appear to change binding potentials and could affect folding conformations of brain-associated structures. Despite the large sequence variation between HIV subtypes, structural alignments confirmed that viral structures derived from patients with HAD were more similar to subtype D structures than to structures derived from patient sequences without HAD. Furthermore, structures derived from brain sequences of patients with HAD were more similar to subtype D structures than they were to their own non-brain structures. The potential finding of a brain-specific nef structure indicates that HAD may result from genetic alterations that alter the folding or binding potential of the protein.     

Differential effects of primary human immunodeficiency virus type 1 nef sequences on downregulation of CD4 and MHC class I

We analyzed two primary nef sequences, KS2 (subtype B) and K306 (subtype D), each directly isolated from patients. Cell lines constitutively expressing respective Nef proteins were constructed using a retroviral vector. There were significant differences in the ability to downregulate surface CD4 and MHC class I proteins between different nef sequences. When the nef sequence from NL432 was used as a reference, KS2 Nef demonstrated the highest ability to downregulate MHC class I, whereas it appeared to lack the ability to downregulate CD4. On the contrary, Nef from K306 decreased the level of surface CD4 to a greater extent, but was less effective on downregulation of MHC class I. These results showed that the levels of downregulation of CD4 and MHC class I could significantly vary among HIV strains and that two well-known functions of Nef, downregulation of CD4 and MHC class I, would be separated.     

Phylogeographic analysis of HIV-1 subtype C dissemination in Southern Brazil

The HIV-1 subtype C has spread efficiently in the southern states of Brazil (Rio Grande do Sul, Santa Catarina and Paraná). Phylogeographic studies indicate that the subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population at some time point between 1960 and 1980, but little is known about the spatial dynamics of viral spread. A total of 135 Brazilian HIV-1 subtype C pol sequences collected from 1992 to 2009 at the three southern state capitals (Porto Alegre, Florianópolis and Curitiba) were analyzed. Maximum-likelihood and Bayesian methods were used to explore the degree of phylogenetic mixing of subtype C sequences from different cities and to reconstruct the geographical pattern of viral spread in this country region. Phylogeographic analyses supported the monophyletic origin of the HIV-1 subtype C clade circulating in southern Brazil and placed the root of that clade in Curitiba (Paraná state). This analysis further suggested that Florianópolis (Santa Catarina state) is an important staging post in the subtype C dissemination displaying high viral migration rates from and to the other cities, while viral flux between Curitiba and Porto Alegre (Rio Grande do Sul state) is very low. We found a positive correlation (r(2) = 0.64) between routine travel and viral migration rates among localities. Despite the intense viral movement, phylogenetic intermixing of subtype C sequences from different Brazilian cities is lower than expected by chance. Notably, a high proportion (67%) of subtype C sequences from Porto Alegre branched within a single local monophyletic sub-cluster. These results suggest that the HIV-1 subtype C epidemic in southern Brazil has been shaped by both frequent viral migration among states and in situ dissemination of local clades.     

Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef

The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.