Performance comparison of the Maxim and Sedia Limiting Antigen Avidity assays for HIV incidence surveillance.

BACKGROUND: Two manufacturers, Maxim Biomedical and Sedia Biosciences Corporation, supply CDC-approved versions of the HIV-1 Limiting Antigen Avidity EIA (LAg) for detecting 'recent' HIV infection in cross-sectional incidence estimation. This study assesses and compares the performance of the two assays for incidence surveillance. METHODS: We ran both assays on a panel of 2,500 well-characterized HIV-1-infected specimens. We analysed concordance of assay results, assessed reproducibility using repeat testing and estimated mean durations of recent infection (MDRIs) and false-recent rates (FRRs) for a range of normalized optical density (ODn) thresholds, alone and in combination with viral load thresholds. We defined three hypothetical surveillance scenarios, similar to the Kenyan and South African epidemics, and a concentrated epidemic. These scenarios allowed us to evaluate the precision of incidence estimates obtained by means of various recent infection testing algorithms (RITAs) based on each of the two assays. RESULTS: The Maxim assay produced lower ODn values than the Sedia assay on average, largely as a result of higher calibrator readings (mean OD of 0.749 vs. 0.643), with correlation of normalized readings lower (R2 = 0.908 vs. R2 = 0.938). Reproducibility on blinded control specimens was slightly better for Maxim. The MDRI of a Maxim-based algorithm at the 'standard' threshold (ODn ≤1.5 & VL >1,000) was 201 days (95% CI: 180,223) and for Sedia 171 (152,191). The difference Differences in MDRI were estimated at 32.7 (22.9,42.8) and 30.9 days (21.7,40.7) for the two algorithms, respectively. Commensurately, the Maxim algorithm had a higher FRR in treatment-naive subjects (1.7% vs. 1.1%). The two assays produced similar precision of incidence estimates in the three surveillance scenarios. CONCLUSIONS: Differences between the assays can be primarily attributed to the calibrators supplied by the manufacturers. Performance for surveillance was extremely similar, although different thresholds were optimal (i.e. produced the lowest variance of incidence estimates) and at any given ODn threshold, different estimates of MDRI and FRR were obtained. The two assays cannot be treated as interchangeable: assay and algorithm-specific performance characteristic estimates must be used for survey planning and incidence estimation.

A lymphomagenic role for HIV beyond immune suppression?

Despite the immune reconstitution promoted by combined antiretroviral therapy (cART), lymphomas still represent the most common type of cancer in HIV-infected individuals. Cofactors related to immunodeficiency such as oncogenic viruses, chronic antigenic stimulation, and cytokine overproduction are thought to be the main drivers of HIV lymphomagenesis, although the current scenario does not convincingly explain the still-high incidence of lymphomas and the occurrence of peculiar lymphoma histotypes in HIV-infected patients under cART. Recent findings are challenging the current view of a mainly indirect role of HIV in lymphoma development and support the possibility that HIV may directly contribute to lymphomagenesis. In fact, mechanisms other than immune suppression involve biologic effects mediated by HIV products that are secreted and accumulate in lymphoid tissues, mainly within lymph node germinal centers. Notably, HIV-infected patients with lymphomas, but not those not affected by these tumors, were recently shown to carry HIV p17 protein variants with enhanced B-cell clonogenic activity. HIV p17 protein variants were characterized by the presence of distinct insertions at the C-terminal region of the protein responsible for a structural destabilization and the acquisition of novel biologic properties. These data are changing the current paradigm assuming that HIV is only indirectly related to lymphomagenesis. Furthermore, these recent findings are consistent with a role of HIV as a critical microenvironmental factor promoting lymphoma development and pave the way for further studies that may lead to the design of more effective strategies for an early identification and improved control of lymphomas in the HIV setting.

A CXCR1 haplotype hampers HIV-1 matrix protein p17 biological activity.

OBJECTIVE: Monocyte inflammatory processes are fundamental events in AIDS pathogenesis. HIV-1 matrix protein p17, released from infected cells, was found to exert an interleukin (IL)-8 chemokine-like activity on human monocytes, promoting their trafficking and sustaining inflammatory processes, after binding to CXCR1. A haplotype of the CXCR1 gene (CXCR1_300_142) has been associated with slow HIV disease progression. Here, we determine how CXCR1 genetic variations impact on p17 biological activity. DESIGN/METHODS/RESULTS: Our results show that Jurkat cells overexpressing CXCR1 or the receptor carrying single polymorphism CXCR1_300 or CXCR1_142 are able to adhere and migrate in response to both IL-8 and p17. On the contrary, Jurkat cells overexpressing CXCR1_300_142 and monocytes of individuals with such CXCR1 polymorphisms lose the capacity to adhere and migrate in response to p17, but not to their physiological ligand IL-8. Surface plasmon resonance (SPR) and multispectral imaging flow cytometry showed that p17 bound with similar affinity to CXCR1 and CXCR1_300_142. Moreover, whereas p17 was able to activate CXCR1, it was incapable of functionally interacting with CXCR1_300_142 by phosphorylating extracellular signal-regulated kinase 1/2, which regulates chemokine-induced cellular responses. Finally, mutagenesis studies showed that, unlike IL-8, p17 does not use Glu-Leu-Arg-like motifs to activate CXCR1. CONCLUSIONS: Our results, showing the inability of p17 to activate CXCR1_300_142, a receptor found to be expressed on immune cells of patients with a low progression of HIV disease, point to a crucial role of p17 in AIDS pathogenesis. Our findings herein call for an exploration of the therapeutic potential of blocking the p17/CXCR1 axis in HIV infection.

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(AU)

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(AU)

HIV-1 matrix protein p17: a candidate antigen for therapeutic vaccines against AIDS.

The success in the development of anti-retroviral therapies (HAART) that contain human immunodeficiency virus type 1 (HIV-1) infection is challenged by the cost of this lifelong therapy and by its toxicity. Immune-based therapeutic strategies that boost the immune response against HIV-1 proteins or protein subunits have been recently proposed to control virus replication in order to provide protection from disease development, reduce virus transmission, and help limit the use of anti-retroviral treatments. HIV-1 matrix protein p17 is a structural protein that is critically involved in most stages of the life cycle of the retrovirus. Besides its well established role in the virus life cycle, increasing evidence suggests that p17 may also be active extracellularly in deregulating biological activities of many different immune cells that are directly or indirectly involved in AIDS pathogenesis. Thus, p17 might represent a promising target for developing a therapeutic vaccine as a contribution to combating AIDS. In this article we review the biological characteristics of HIV-1 matrix protein p17 and we describe why a synthetic peptide representative of the p17 functional epitope may work as a vaccine molecule capable of inducing anti-p17 neutralizing response against p17 derived from divergent HIV-1 strains.

Early immune senescence in HIV disease.

Non-AIDS-defining co-morbidities that occur despite viral suppression and immune reconstitution using antiretroviral therapy depict early aging process in HIV-infected individuals. During aging, a reduction in T-cell renewal, together with a progressive enrichment of terminally differentiated T cells, translates into a general decline of the immune system, gradually leading to immunosenescence. Inflammation is a hallmark of age-associated comorbidities, and immune activation is a hallmark of HIV disease. Constant stimulation of the immune system by HIV or due to co-infections activates the innate and adaptive immune system, resulting in release of mediators of inflammation. Immune activation coupled with lack of anti-inflammatory responses likely results in accelerated aging in HIV disease. Dysfunctional thymic output, along with HIV-mediated disruption of the gastrointestinal barrier leading to microbial translocation, contributes to the circulating antigenic load driving early senescence in HIV disease.

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.

HIV-1 matrix protein: a mysterious regulator of the viral life cycle.

Significant progress has been achieved in the last few years concerning the human immunodeficiency virus (HIV-1) life cycle, mostly in the fields of cellular receptors for the virus, virus assembly and budding of virus particles from the cell surface. Meanwhile, some aspects, such as postentry events, virus maturation and the regulatory role of individual viral proteins remain poorly defined. This review summarizes some recent findings concerning the role of Gag Pr55 and its proteolytic processing in the HIV-1 life cycle with particular emphasis on the functions of matrix protein p17 (MA), the protein which plays a key role in regulation of the early and late steps of viral morphogenesis. Based on our recent observations, the possibility is discussed that two subsets of MA exist, one cleaved from the Gag precursor in the host cell (cMA), and the other cleaved in the virions (vMA). It is suggested that two MA fractions possess diverse functions and are involved in different stages of virus morphogenesis as key regulators of the viral life cycle.

Inhibition of viral assembly in murine cells by HIV-1 matrix.

In human cells, the N-terminal matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag targets assembly to specific membrane compartments. In murine fibroblasts, membrane targeting of Gag and assembly of HIV-1 are inefficient. These deficiencies are relieved by replacement of HIV-1 MA with murine leukemia virus (MLV) MA in chimeric proviruses. In this study, we examined chimeric HIV-1 carrying tandem MLV and HIV-1 MA domains and found that the addition of MLV MA to the N-terminus of HIV-1 Gag enhanced membrane binding in murine cells, but was not sufficient to stimulate virus production. Removal of HIV MA was required to observe more efficient Gag processing and increased virus production in murine cells. Deletion of the globular head of MA also alleviated the blocks to membrane binding and Gag processing in murine cells, yet did not lead to increased virus production. These MA-dependent, cell-type-specific phenotypes suggest that host factors interact with the globular head of MA to regulate membrane binding and additional membrane-independent step(s) required for assembly.

Functions of the HIV-1 matrix protein p17.

HIV-1 replication is a dynamic process influenced by a combination of viral and host factors. The HIV-1 matrix protein p17 is a structural protein critically involved in most stages of the life cycle of the retrovirus. It participates in the early stages of virus replication as well as in RNA targeting to the plasma membrane, incorporation of the envelope into virions and particle assembly. Besides its well established functions, p17 acts as a viral cytokine that works on preactivated--but not on resting--human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication after binding to a cellular receptor (p17R). Thus, p17 might play a key role in the complex network of host- and virus-derived stimulatory factors contributing to create a favourable environment for HIV-1 infection and replication. Here, we present a brief overview of the functions played by the matrix protein p17 in the HIV-1 life cycle and summarize the current understanding of how p17 could contribute to the pathogenesis of HIV-1 disease.

Human immunodeficiency virus type 1 KK26-27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import.

We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26-27 in MA reported nuclear localization signal (NLS)-1 impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.

Diversity of mosaic structures and common ancestry of human immunodeficiency virus type 1 BF intersubtype recombinant viruses from Argentina revealed by analysis of near full-length genome sequences.

The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.

Regulation of class II expression in monocytic cells after HIV-1 infection.

Human macrophage hybridoma cells were used to study HLA-DR expression after HIV-1 infection. HLA-DR surface expression was lost 2 wk after infection that was associated with decreased mRNA transcription. Transfecting HLA-DR-alpha and HLA-DR-beta cDNA driven by a nonphysiological CMV promoter restored expression, suggesting that regulatory DNA-binding proteins may be affected by HIV-1 infection. There was no protein binding to conserved class II DNA elements (W/Z/S box, X-1 and X-2 boxes, and Y box) in a HIV-1-infected human macrophage hybridoma cell line, 43(HIV), and in primary monocytes that lost HLA-DR expression after HIV-1(BaL) infection. PCR analysis of the HIV-1-infected cells that lost HLA-DR expression revealed mRNA for W/Z/S (RFX-5), X-1 (RFX-5), X-2 (hX-2BP), and one Y box DNA-binding protein (NF-YB), and CIITA, a non-DNA-binding protein necessary for class II transcription. There was no mRNA for the Y box-binding protein, NF-YA. However, HLA-DR expression could be restored by transfection with NF-YA driven by a CMV promoter, although HLA-DR failed to localize in either the late endosomes, lysosomes, or acidic compartments. This was associated with a loss of class II-associated invariant chain peptide and leupeptin-induced protein in the 43(HIV) cells. To address this further, non-HIV-1-infected 43 cells were infected with vaccinia virus containing HIV-1 gag, nef, pol, and env proteins. HLA-DR failed to localize in neither the late endosomes, lysosomes, or acidic compartments in the vaccinia-infected cells containing HIV-1 env protein. HIV-1 appears to have multiple effects on class II expression in monocytic cells that may contribute to the immune defects seen in HIV-1-infected patients.

Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells.

We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.

A human nuclear shuttling protein that interacts with human immunodeficiency virus type 1 matrix is packaged into virions.

Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55(Gag) and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4(+) T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55(Gag) and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus.

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.

Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages.

Human immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17(Gag)) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7. deltaVpr.deltaNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.deltaVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.deltaVpr and R7.deltaVpr.deltaNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection.

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.

Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17.

The human immunodeficiency virus type 1 (HIV-1) matrix protein, p17, plays important roles in both the early and late stages of the viral life cycle. Using our previously determined solution structure of p17, we have undertaken a rational mutagenesis program aimed at mapping structure-function relationships within the molecule. Amino acids hypothesized to be important for p17 function were mutated and examined for effect in an infectious proviral clone of HIV-1. In parallel, we analyzed by nuclear magnetic resonance spectroscopy the structure of recombinant p17 protein containing such substitutions. These analyses identified three classes of mutants that were defective in viral replication: (i) proteins containing substitutions at internal residues that grossly distorted the structure of recombinant p17 and prevented viral particle formation, (ii) mutations at putative p17 trimer interfaces that allowed correct folding of recombinant protein but produced virus that was defective in particle assembly, and (iii) substitution of basic residues in helix A that caused some relocation of virus assembly to intracellular locations and produced normally budded virions that were completely noninfectious.