A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Absorbance summation: A novel approach for analyzing high-throughput ELISA data in the absence of a standard.

We have developed a very simple method, termed absorbance summation (AS), for comparing protein concentrations between samples in ELISA assays without a standard. This method sums the observed absorbance values from all dilutions to obtain one data point for each sample to be used for comparison. AS is less computationally intensive than fitting sigmoidal curves, and it avoids the difficulty of parameter estimation for samples with absorbance values lying primarily at the lower tail of the curve. Our simulation studies showed that it performs much better than the sigmoidal curve fitting method and the conventional endpoint titer method. The power of this simple method is as high as the formal curve fitting followed by the estimation of area under the curve (AUC).

The Role of IL-27 and its Receptor in the Pathogenesis of HIV/AIDS and Anti-viral Immune Response.

BACKGROUND: Cytokines have been widely demonstrated to involve in the pathogenesis of AIDS and the mechanisms of antiretroviral therapy. Interleukin 27 (IL-27) is a new member of the IL-12 cytokine family and has been shown to interfere HIV-1 virus replication with controversial findings. This study is to investigate the dynamic changes in plasma IL-27 level and cell surface IL-27 receptor expression in HIV/AIDS patients who underwent HAART. METHODS: Whole blood was collected from 34 HIV-positive/AIDS patients 0, 6, and 12 months after initiation of HAART and 27 healthy subjects. Plasma IL-27, IFN-γ, and IL-4 were measured by enzyme-linked immunosorbent assay, while peripheral blood CD3+CD4+ T cells count and the gp130 expressed CD3+CD4+cell were measured by flow cytometry. RESULTS: The plasma IL-27 concentration, IFN-γ concentration, and percentage of positive gp130 CD4 cells were significantly decreased in previously treatment-naive HIV/AIDS patients compared to healthy controls, but gradually increased 6 and 12 months after initiation of HAART. Conversely, IL-4 levels were significantly increased in treatment-naive HIV/AIDS patients compared to healthy controls, but gradually decreased 6 and 12 months after HAART. The concentrations of plasma IL-27 were positively correlated with the percentage of gp130 positive CD4 cells (r=0.438, p=0.016). Both plasma IL-27 concentration and gp130 positive cell percentage were positively associated with peripheral blood CD3+CD4+ T cell count (P<0.05 or P<0.01), but negatively associated with plasma HIV viral load (P<0.05 or P<0.01). CONCLUSION: IL-27 signaling (IL-27 and its receptor) may be involved in the pathogenesis of HIV infection and immune reconstitution in HIV/AIDS patients who underwent HAART. IL-27 may exert effects through regulating Th1 / Th2 ratio.

Quantifying CD4/CCR5 Usage Efficiency of HIV-1 Env Using the Affinofile System.

Entry of HIV-1 into target cells involves the interaction of the HIV envelope (Env) with both a primary receptor (CD4) and a coreceptor (CXCR4 or CCR5). The relative efficiency with which a particular Env uses these receptors is a major component of cellular tropism in the context of entry and is related to a variety of pathological Env phenotypes (Chikere et al. Virology 435:81-91, 2013). The protocols outlined in this chapter describe the use of the Affinofile system, a 293-based dual-inducible cell line that expresses up to 25 distinct combinations of CD4 and CCR5, as well as the associated Viral Entry Receptor Sensitivity Assay (VERSA) metrics used to summarize the CD4/CCR5-dependent infectivity results. This system allows for high-resolution profiling of CD4 and CCR5 usage efficiency in the context of unique viral phenotypes.

Nef Proteins from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity.

UNLABELLED: Impairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers [EC]). Elimination of HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype. IMPORTANCE: Nef clones derived from elite controllers (EC) have been shown to be attenuated for CD4 downregulation; how this contributes to the nonprogressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody-dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here, we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nef's ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.

Glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV-1 gp140 trimers in 293T cells identifies disulfide heterogeneity present in both proteins and differences in O-linked glycosylation.

The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env's PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens.

Detection and initial characterization of protein entities consisting of the HIV glycoprotein cytoplasmic C-terminal domain alone.

Employing antibodies against the cytoplasmic tail of the HIV-1 glycoprotein (Env-CT), in addition to gp160/gp41, we have identified several novel small Env proteins (<25kD) in HIV-1 transfected and infected cells. Mass spectrometric and mutational analyses show that two mechanisms contribute to their generation. Thus the protein, designated Tr-Env-CT (for truncated Env-CT), consists of the C-terminal 139 amino acids (aa) of Env (aa 718-856) with the N-terminal Q718 modified to pyroglutamic acid. It is likely derived from full-length Env protein by proteolytic processing. A further heterogeneous set of slightly larger proteins, termed Env-CT* species, are rather derived from spliced mRNAs containing only those Env C-terminal residues (aa 719-856) which overlap with the second tat and rev coding exons. They are N-terminally extended in the same reading frame. It is conceivable that essential Env-CT functions may be fulfilled by these novel species rather than by the full-length glycoprotein itself.

Evaluación de la inmovilización de un péptido de gp36 de VIH-2 en membranas de nitrocelulosa / Evaluation of the immobilization of one HIV-2 gp36 peptide in nitrocellulose membrane

Introducción: la inmovilización de antígenos a soportes sólidos se utiliza para el desarrollo de diversos inmunoensayos. Una de las primeras tecnologías desarrolladas fue la adsorción de proteínas por aplicación directa sobre la nitrocelulosa. Objetivo: normalizar la inmovilización de un péptido sintético de la proteína de transmembrana gp36 del VIH-2, a un soporte de nitrocelulosa para fines diagnósticos y evaluar los parámetros de desempeño en un grupo de muestras de sueros con reactividad de interés conocida. Métodos: el péptido se inmovilizó de forma libre, conjugado a la albúmina de suero bovina (BSA) y a la hemocianina de lapa marina (KLH) como proteínas portadoras. Se analizaron los parámetros de inmovilización y se determinó la variante óptima. Con la variante escogida se evaluó la sensibilidad y especificidad diagnóstica frente a paneles de referencia del Laboratorio de Investigaciones del SIDA. La especificidad analítica se evaluó con muestras reactivas a VIH-1 y HTLV-I. Resultados: el análisis de las variantes de péptido inmovilizadas a las membranas de nitrocelulosa, demostró que el péptido gp36-BSA, fue el que logró la mayor diferenciación entre muestras positivas y negativas. Se obtuvo 100 por ciento de sensibilidad y 95,2 por ciento de especificidad diagnóstica, así como 100 por ciento de especificidad analítica. Conclusiones: el péptido gp36-BSA inmovilizado en membranas de nitrocelulosa es eficaz en el diagnóstico serológico del VIH-2, lo cual permitirá considerarlo para su empleo con fines diagnósticos en sistemas que utilicen como fase sólida la nitrocelulosa.

Introduction: antigen immobilization in solid supports is used for the development of several immunoassays. One of the first technologies developed was the protein adsorption by direct application to nitrocellulose. Objective: to standardize the immobilization of a synthetic peptide of the HIV-2 transmembrane protein gp36 to nitrocellulose support for diagnostic purposes and to evaluate the performance parameters in a group of serum samples with recognized interesting reactivity. Methods: the peptide was freely immobilized, conjugated to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) as carrier proteins. Immobilization parameters were analyzed and then, the optimal immobilization alternative was determined. Using the chosen variant, the diagnostic sensitivity and specificity against reference panels of the AIDS Research Laboratory were evaluated. Analytical specificity was evaluated with reactive samples to HIV-1 and HTLV-I. Results: the analysis of the immobilized peptide variants to nitrocellulose membranes showed that the gp36 peptide-BSA was the one that succeeded in setting the greatest differentiation between positive and negative samples. There were observed 100 percent sensitivity, 95.2 percent diagnostic specificity and 100 percent analytical specificity. Conclusions: the gp36-BSA peptide immobilized on nitrocellulose membranes showed efficacy for the serological diagnosis of HIV-2, which will allow considering this peptide for diagnostic uses in systems with nitrocellulose based solid phase.

Características virológicas del VIH / Virological characteristics of HIV

El virus de la inmunodeficiencia humana tipo 1 (VIH-1) es el agente productor del sida una enfermedad reconocida desde hace 30 años que ha alcanzado proporciones pandémicas. Su origen se remonta a la transmisión a humanos de retrovirus que infectan a poblaciones de chimpancés en África central hace aproximadamente 100 años. Desde esta localización su expansión a todo el mundo ha sido espectacular principalmente en las últimas décadas. La intensa investigación realizada nos permite disponer de un tratamiento eficaz para controlar la replicación del virus y evitar la progresión de la enfermedad sin embargo no disponemos aún de una vacuna que impida la continua extensión de la pandemia. No es posible entender estos fenómenos sin un conocimiento detallado de la biología del VIH-1 y los mecanismos que se han seleccionado en este asombroso agente para infectar una célula clave como el linfocito T CD4+ y evadir la respuesta inmune (AU)

The human immunodeficiency virus type 1 (HIV-1) is the agent that causes AIDS, a disease known for 30 years that has reached pandemic proportions. Its origin dates back to human transmission of retroviruses infecting populations of chimpanzees in central Africa about 100 years ago. From this location its expansion to the whole world has been phenomenal, particularly in recent decades. Extensive research has led to an effective treatment for controlling virus replication and to prevent progression of the disease, but we do not yet have a vaccine to prevent the continuing spread of the pandemic. It is not possible to understand these phenomena without detailed knowledge of the biology of HIV-1 and the mechanisms that have been selected in this amazing agent to infect a key cell such as the CD4 + T cell and evade the immune response (AU)

[Evaluation of the immobilization of one HIV-2 gp36 peptide in nitrocellulose membrane]. / Evaluación de la inmovilización de un péptido de gp36 de VIH-2 en membranas de nitrocelulosa.

INTRODUCTION: antigen immobilization in solid supports is used for the development of several immunoassays. One of the first technologies developed was the protein adsorption by direct application to nitrocellulose. OBJECTIVE: to standardize the immobilization of a synthetic peptide of the HIV-2 transmembrane protein gp36 to nitrocellulose support for diagnostic purposes and to evaluate the performance parameters in a group of serum samples with recognized interesting reactivity. METHODS: the peptide was freely immobilized, conjugated to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) as carrier proteins. Immobilization parameters were analyzed and then, the optimal immobilization alternative was determined. Using the chosen variant, the diagnostic sensitivity and specificity against reference panels of the AIDS Research Laboratory were evaluated. Analytical specificity was evaluated with reactive samples to HIV-1 and HTLV-1. RESULTS: the analysis of the immobilized peptide variants to nitrocellulose membranes showed that the gp36 peptide-BSA was the one that succeeded in setting the greatest differentiation between positive and negative samples. There were observed 100 % sensitivity, 95.2 % diagnostic specificity and 100 % analytical specificity. CONCLUSIONS: the gp36-BSA peptide immobilized on nitrocellulose membranes showed efficacy for the serological diagnosis of HIV-2, which will allow considering this peptide for diagnostic uses in systems with nitrocellulose -based solid phase.

Short communication: Human blood dendritic cells are infected separately from monocytes in HIV type 1 patients.

Monocytes serve as a systemic reservoir of myeloid precursors for the renewal of tissue macrophages and dendritic cells (DCs). Both monocytes and dendritic cells can be infected with HIV-1. Circulating DCs are believed to be derived from a common precursor of monocytes, or, in the case of inflammatory challenge, from monocytes directly. Because there are fewer infected blood monocytes than infected cells after differentiation, we hypothesized that the majority of HIV-1 infection in circulating DCs occurs via direct viral binding to their CD4 and coreceptors after differentiation. We isolated monocytes at one time point and circulating dendritic cells at a second time point from the blood of HIV-1-infected patients. Proviral DNA was isolated from DCs and monocytes, and the C2-V4 region of the HIV-1 env gene was cloned and sequenced. Phylogeny, nucleotide distances, and glycosylation patterns of the env gene were performed. The phylogenetic trees revealed that viral forms from the monocytes clustered distantly from the quasispecies derived from circulating DCs. The nucleotide distances and differing glycosylation patterns suggest that the infection of DCs is independent of the infection of the monocytes.

Young pregnant women living with HIV/AIDS in Criciuma, Southern Brazil, are infected almost exclusively with HIV type 1 clade C.

Southern Brazil has the highest prevalence rate of AIDS in the country and is the only region in the Americas where HIV-1 C prevails. Metropolitan areas and harbor cities have been evaluated, but limited information is available for small towns and specific populations. We studied women attending the obstetric outpatient clinic of Criciuma, State of Santa Catarina in 2007 to evaluate the molecular epidemiology of HIV-1 among pregnant women living with HIV/AIDS. Forty-two cases had partial pol gene sequenced and additional partial gag and/or env genes from nine women. HIV subtyping was evaluated by phylogenetic methods and antiretroviral (ARV) drug resistance mutations (DRMs) at the Stanford Database. DRMs to one or more ARV class was observed in 20/42, 48% of cases, with 15/41, 37% with viral load <500 copies/ml. Subtype C at pol was identified in 33/42, 78.6% (95% CI: 64-89%), C mosaics (CB, CF) in 2, 4.8% (95% CI: 0.8-19%), F in 4, 9.5% (95% CI: 3-21%), and B in 3, 7.1% (95% CI: 1.8-18%). Discordance in concatenated gag/pol/env or intraregion mosaic was observed in 1/9, 11% of HIV-1 C genomes. The proportion of HIV-1 C in this study is the highest rate described in the Americas. Molecular surveillance in specific populations is instrumental for a better understanding of the Brazilian HIV epidemic.

Genetic characterization of eight full-length HIV type 1 genomes from the Democratic Republic of Congo (DRC) reveal a new subsubtype, A5, in the A radiation that predominates in the recombinant structure of CRF26_A5U.

In this study, we characterized HIV-1 strains from the Democratic Republic of Congo (DRC), previously described as divergent subtype A (n = 1, 97CD.KMST91) or untypable (n = 7) in the V3-V5 env region. Four strains had the same structure over the entire genome, including alternating fragments of a new subsubtype, A5, within the subtype A radiation and fragments that remain unclassified. Therefore, the cluster of new viruses represents a new circulating recombinant, CRF26_A5U. Three additional strains were unique recombinants with the newly described CRF26_A5U and subtype C. Finally, the nearly full-length sequence of 97CD.KMST91 showed that this strain also consisted of alternating fragments of a divergent subtype A lineage and unclassified fragments, although different from previously reported A and U sequences. The high genetic distances among the different CRF26-A5U strains suggest their longstanding presence in the DRC.

Comparison of Bayesian and maximum-likelihood phylogenetic approaches in two legal cases involving accusations of transmission of HIV.

We investigated two cases of alleged criminal transmission of HIV-1 using Bayesian and maximum-likelihood phylogenetic approaches to determine whether the inference method used influenced the outcome in these cases. In the first case, Bayesian methods were used to reexamine gag and env sequences from an earlier investigation in which the HIV-1 strains infecting one of several contacts could not be linked phylogenetically to that of the accused despite strongly suggestive epidemiological evidence. In the second case, maximum-likelihood and Bayesian inference methods were used to investigate the relatedness of gag and env sequences from HIV-1 strains infecting a man accused of intentionally transmitting the virus to several contacts. Bayesian analysis of HIV-1 strains from the first case confirmed earlier results obtained by maximum-likelihood analysis. A monophyletic cluster linking viruses from the accused and three of his direct and indirect contacts was supported, but a linkage between these viruses and a fourth epidemiologically linked contact could not be demonstrated. In the second case, a strong virological link between the accused and two of his contacts, and the absence of links with four other contacts, was confirmed by both maximum-likelihood and Bayesian inference methods. It is important that phylogenetic programs applied in a legal setting are conservative in their outcome. Although Bayesian methods offer computational tractability for large data sets and complex evolutionary models, this study demonstrates they do not assist when clear linkages between viruses are demonstrated using maximum-likelihood methods.

Multiple-infection and recombination in HIV-1 within a longitudinal cohort of women.

BACKGROUND: Recombination between strains of HIV-1 only occurs in individuals with multiple infections, and the incidence of recombinant forms implies that multiple infection is common. Most direct studies indicate that multiple infection is rare. We determined the rate of multiple infection in a longitudinal study of 58 HIV-1 positive participants from The Women's Interagency HIV Study with a richer sampling design than previous direct studies, and we investigated the role of recombination and sampling design on estimating the multiple infection rate. RESULTS: 40% of our sample had multiple HIV-1 infections. This rate of multiple infection is statistically consistent with previous studies once differences in sampling design are taken into account. Injection drug use significantly increased the incidence of multiple infections. In general there was rapid elimination of secondary strains to undetectable levels, but in 3 cases a superinfecting strain displaced the initial infecting strain and in two cases the strains coexisted throughout the study. All but one secondary strain was detected as an inter- and/or intra-genic recombinant. Injection drug use significantly increased the rate of observed recombinants. CONCLUSION: Our multiple infection rate is consistent with rates estimated from the frequency of recombinant forms of HIV-1. The fact that our results are also consistent with previous direct studies that had reported a much lower rate illustrates the critical role of sampling design in estimating this rate. Multiple infection and recombination significantly add to the genetic diversity of HIV-1 and its evolutionary potential, and injection drug use significantly increases both.

The infectious molecular clone and pseudotyped virus models of human immunodeficiency virus type 1 exhibit significant differences in virion composition with only moderate differences in infectivity and inhibition sensitivity.

Two frequently employed methods for generating well-characterized, genetically defined infectious human immunodeficiency virus type 1 in vitro include the use of infectious molecular clones (IMCs) and pseudoviruses (PVs) competent for single-round infection. We compared six matched pairs of IMCs and PVs. The relative amounts of Env incorporated and efficiency of cleavage differed substantially between the two systems. Altering the ratio of proviral genome and env expression plasmids can produce pseudovirions that are structurally more similar to the matched IMCs. Differences in Env incorporation and cleavage translated into moderate differences in assays infectivity and sensitivity to neutralizing antibodies and entry inhibitors.

Maximizing coverage of glycosylation heterogeneity in MALDI-MS analysis of glycoproteins with up to 27 glycosylation sites.

Glycosylation affects various biological functions of proteins (e.g., protein binding, inter- or intracell signaling, etc.), and it can serve as an indicator of disease. Therefore, characterization of the glycosylation in proteins is one important step in developing a comprehensive understanding of the biological significance of glycosylation and in facilitating disease diagnosis. Glycopeptide-based MS analysis has proven to be a viable tool for glycopeptide analysis. However, when glycopeptides coexist with peptides, glycopeptide signals are usually suppressed by the strongly ionizing peptides. Toward this end, it would be desirable to seek methods to improve glycopeptide detection. Herein, we performed an in-depth study of maximizing glycosylation coverage on model glycoproteins by optimizing all the aspects of glycopeptide-based analysis, including sample preparation methods, mass spectral techniques, and data analysis strategies. For sample preparation, several approaches, including reversed-phase high-performance liquid chromatography, lectin-based affinity, and hydrophilic affinity using a carbohydrate-based resin, were compared and tested individually as well as in parallel. For mass spectral techniques, profiling glycopeptides in both positive and negative ion mode is essential to obtain complete glycan profiles. For data analysis, incorporating variable modifications in the database search of GlycoPep DB enhances glycopeptide coverage. In addition, the use of PNGase F helps to confirm the presence of weakly ionized glycopeptides when they coelute with strongly ionizing species. In doing so, we created a work flow that is designed specifically to optimize the coverage of glycosylation heterogeneity in terms of the number of glycosylation sites detected and their corresponding glycan profiles. To test the effectiveness of this approach, a glycoprotein with 27 potential glycosylation sites, and an unknown glycosylation profile, was analyzed; on this protein, more than 300 glycoforms from 23 detected glycosylation sites were identified. This work demonstrates that these strategies significantly improve the glycopeptide detection, thereby, facilitating understanding the functional properties of glycans on the glycoproteins.

HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes.

Several viruses encode ion channels that both modulate the trafficking of envelope glycoprotein(s) and stimulate the release of virions from cells. HIV-1 Vpu enhances virion release and inhibits the endosomal accumulation of the viral structural protein Gag. We investigated whether Vpu affects the subcellular distribution of Env as well as Gag. Env and Vpu colocalized with each other, in part within the trans-Golgi network. In the absence of Vpu, Env accumulated more extensively within clathrin-coated endosomal structures. These structures had several features consistent with an endosomal viral assembly domain: they contained Gag, including proteolytically processed viral matrix protein; the tetraspanins CD63 and CD81; the adaptor protein complex AP-3; and AIP1/ALIX, a cellular cofactor for viral budding. These endosomes labelled incompletely with Env derived from the cell surface, suggesting that some Env reaches this compartment without transiting the plasma membrane. Consistent with this, endosomal accumulation of Env was not blocked by dominant-negative Eps15, an inhibitor of AP-2-mediated endocytosis. Although these data are potentially explained by greater endocytosis of mature virions in the absence of Vpu, they also raise the possibility that Vpu inhibits the transport of Env and Gag to late endosomes, leading to viral assembly at the plasma membrane.

Passive immunization as tool to identify protective HIV-1 Env epitopes.

The HIV-1/AIDS epidemic continues to escalate, and a protective vaccine remains elusive. The first vaccine candidate, gp120, did not induce broadly neutralizing antibodies (nAbs) against primary HIV-1 isolates and was ineffective in phase III clinical trials. Attention then focused on generating cytotoxic lymphocyte (CTL)-based vaccines. Interest in anti-HIV-1 nAbs was renewed when passive immunization with human neutralizing monoclonal antibodies (nmAbs) completely protected macaques after intravenous and mucosal challenges with simian-human immunodeficiency viruses (SHIVs) encoding HIV-1 env. These nmAbs targeted conserved, functionally important epitopes on gp120 and gp41. Protection in primate/SHIV models was observed when nmAbs were used singly (nmAbs 2G12, b12) and in various combination regimens (nmAbs b12, F105, 2G12, 2F5, 4E10). Passive immunization, a well-established tool to determine the correlates of protective immunity, thus identified protective epitopes. The three-dimensional structures of some of the latter were recently elucidated, generating important information to design nAb-response-base immunogens. However, several of the protective nmAbs were found to exhibit autoreactivity, raising the possibility that B-cell responses against the cognate epitopes may be difficult to induce by active immunization. It will be important to explore whether broad neutralization can be dissociated from autoreactivity. Future experiments will reveal whether other conserved HIV-1 Env epitopes exist, antibodies against which will be broadly neutralizing in vitro, protective as passive immunization in SHIV-challenged macaques, but lacking autoreactivity. Since all protective epitopes identified to date are located on HIV-1 clade B Env, future studies should include analysis of nmAbs against non-clade B strains.

Characterization of replication defects induced by mutations in the basic domain and C-terminus of HIV-1 matrix.

Extensive mutagenesis has defined distinct functional domains in the HIV-1 matrix domain (MA). In an attempt to more clearly define functions of regions of MA which affect viral entry, we analyzed mutations in the N-terminal basic and the C-terminal helical domains. Deletions of 8-10 amino acid residues of the C-terminal fifth helix of MA resulted in viruses that were only mildly defective in infectivity and fusion. The defect exhibited by these mutations could largely be attributed to a reduction in levels of viral envelope incorporated into mature virions. Truncation of the gp41 cytoplasmic tail (gp41CT) could rescue the phenotype of one of these mutants. In contrast, mutations of multiple basic residues in the N-terminus of MA were severely defective in both infectivity and fusion. While these mutations induce severe envelope incorporation defects, they also result in virus crippled at a post-entry step, since truncation of the gp41CT could not rescue the infectivity defect.