Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

The role of nucleocapsid of HIV-1 in virus assembly

The role of the nucleocapsid protein of HIV-1 Gag in virus assembly was investigated using Gag truncation mutants, a nucleocapsid deletion mutant, and point mutations in the nucleocapsid region of Gag, in transfected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid regions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild-type HIV-1. A deletion mutant lacking p7 nucleocapsid but containing the C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the first zinc finger of p7 could assemble more efficiently into virions. These results clearly show that p7 is indispensable for virus assembly and release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been previously shown to have reduced RNA binding in vitro [Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996). J. Virol. 70: 771-777], were shown to reduce virus assembly dramatically when expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to have nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994) J. Biol. Chem. 269: 28420-28428] released pelletable material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. These results demonstrate the essential role of HIV-1 nucleocapsid in the virus assembly process and show that the positively charged N terminus of p7 is critical for this role.     

Polymorphism of bacteriophage T7

For viruses made of nucleic acid and protein, the structure of the protein outer shell has, in the past, been found to be uniquely determined by the viral genome. However, here, non-denaturing agarose gel electrophoresis of bacteriophage T7 reveals two states of the mature T7 capsid; the conditions of growth are found to alter the population by T7 of these two electrophoretically defined states. Both states have been previously observed for a genetically altered T7 and they are observed here for wild-type T7. The average electrical surface charge density of a bacteriophage particle (delta) determines its state; the delta of particles in both states is negative. For a given condition of growth, the population of these two states is influenced by the extent to which the major T7 outer shell protein, p10A, is accompanied by its minor readthrough variant, p10B. Comparison of the two electrophoretic states reveals the following. (1) No difference in radius is present in the outer shell (+/-2%). (2) As the pH of electrophoresis is either increased or decreased from neutrality, the state becomes more highly populated for which delta is greater in magnitude (state 1). By changing the pH, some T7 particles are made to change state. (3) Particles in state 1 adsorb less quickly to host cells than do the particles in the alternative state (state 2). This latter observation suggests the hypothesis that state 1 evolved to reduce the probability of re-initiating an infection when conditions are not favorable for growth. This hypothesis is supported by the observation that, as conditions of growth become apparently more unfavorable, progeny increasingly populate state 1.     

Conservation of cytotoxic T lymphocyte (CTL) epitopes as a host strategy to constrain parasite adaptation: evidence from the nef gene of human immunodeficiency virus 1 (HIV-1)

Host cytotoxic T lymphocytes (CTLs) that recognize specific viral peptides (epitopes) are thought to provide the most effective control of viral replication and spread. However, viruses may escape this recognition through mutations in CTL epitopes. We tested the hypothesis that, as an adaptation on the part of the host to constrain parasite escape from immune control, class I major histocompatibility complex (MHC) molecules present peptides that are derived from conserved regions of foreign proteins to CTLs. We did this by estimating the relative conservation of CTL epitopes of the functionally important Nef protein of human immunodeficiency virus 1 (HIV-1) and relating this to the structure and function of the protein. In comparisons among sequences from several HIV-1 subtypes and both major groups, CTL epitopes had lower rates of nonsynonymous nucleotide substitution per site than did the remainder of the protein, indicating the relative conservation of these epitopes. In contrast, helper T-cell epitopes were as conserved as, and monoclonal antibody epitopes less conserved than, the remainder of the protein. The conservation of CTL epitopes is apparently due to their derivation from functionally important domains of Nef, since CTL epitopes coincide with these domains and these domains are conserved relative to the remainder of the protein, in contrast to secondary structural elements, which are not. Recent studies provide evidence of CTL selection on HIV-1 epitopes, but the variational range of viral escape mutants appears to be limited by functional constraints on the protein regions from which epitopes are derived. The presentation of conserved foreign peptides to CTLs by class I MHC molecules may be a general adaptation of vertebrate hosts to constrain the adaptation of their intracellular parasites.     

Neutralizing antibodies from the sera of human immunodeficiency virus type 1-infected individuals bind to monomeric gp120 and oligomeric gp140

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.     

Immunization with hybrid hepatitis B virus core particles carrying circumsporozoite antigen epitopes protects mice against Plasmodium yoelii challenge

The hepatitis B virus nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for circumsporozoite protein (CS) repeat B cell epitopes of the rodent malaria agent Plasmodium yoelii. A vector expressing a hybrid gene coding for the dominant CS repeat epitope (QGPGAP)4 was constructed and transformed into avirulent Salmonella typhimurium. The resulting hybrid HBcAg-CS polyproteins were purified from recombinant Salmonella typhimurium. They purified as particles and displayed HBc as well as P. yoelii CS antigenicity. To investigate immunogenicity and protective efficacy, BALB/c mice were immunized with the hybrid HBcAg-CS particles. Immunization resulted in high titered antinative CS serum IgG antibody litres. BALB/c mice immunized with hybrid HBcAgCS particles were between 90-100% protected against subsequent P. yoelli challenge. Protective immunity persisted for a minimum of three months. These data confirm the previous suggestion (Sch?del et al., 1994), that hybrid HBcAg particles could become a useful component of future human malaria vaccines.     

Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.     

Particle size determinants in the human immunodeficiency virus type 1 Gag protein

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.     

B-cell epitopes in HIV-1 Tat and Rev proteins colocalize with T-cell epitopes and with functional domains

We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.     

A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE

We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17.     

Display of functional alphabeta single-chain T-cell receptor molecules on the surface of bacteriophage

The ability to display functional T-cell receptors (TCR) on the surface of bacteriophage could have numerous applications. For instance, TCR phage-display could be used to develop new strategies for isolating TCRs with unique specificity or it could be used to carry out mutagenesis studies on TCR molecules for analyzing their structure-function. We initially selected a TCR from the murine T-cell hybridoma, DO11.10, as our model system, and genetically engineered a three domain single-chain TCR (scTCR) linked to the gene p8 protein of the Escherichia coli bacteriophage fd. Immunoblotting studies revealed that (1) E. coli produced a soluble scTCR/p8 fusion protein and (2) the fusion protein was packaged by the phage. Cellular competition assays were performed to evaluate the functionality of the TCR and showed the DO11.10 TCR-bearing phage could significantly inhibit stimulation of DO11.10 T hybridoma cells by competing for binding to immobilized MHC/peptide IA(d)/OVA(323-339). Flow cytometric analysis was carried out to evaluate direct binding of DO11.10 TCR-bearing phage onto the surface of cells displaying either IAd containing irrelevant peptide or OVA peptide. The results revealed binding of DO11.10 TCR-bearing phage only on cells expressing IA(d) loaded with OVA peptide showing TCR fine specificity for peptide. To illustrate the generality of TCR phage-display, we also cloned and displayed on phage a second TCR which recognizes a peptide fragment from human tumor suppressor protein p53 restricted by HLA-A2. These findings demonstrate functional TCR can be displayed on bacteriophage potentially leading to the development of novel applications involving TCR phage-display.     

Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load

Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.     

Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization

We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.     

Potent neutralization of primary HIV-1 isolates by antibodies directed against epitopes present in the V1/V2 domain of HIV-1 gp120

Although it is known that some human immune sera possess potent neutralizing activities for primary viruses, the identity of the target epitopes mediating this neutralization is unknown, and currently available immunogens have not been able to induce such activities. Using recombinant fusion glycoproteins expressing native V1/V2 domains of gp120 we have found that sera from a subset of HIV-1-infected humans contain antibodies that recognize broadly conserved V1/V2 epitopes. Such antibodies were isolated from one human serum by affinity chromatography on a column containing a V1/V2 fusion protein, and shown to efficiently neutralize several macrophage-tropic HIV-1 isolates. Rodents immunized with the purified V1/V2 fusion protein produced antibodies reactive with unrelated V1/V2 fusion proteins and with heterologous gp120s. V1/V2-specific immunoglobulins isolated from sera of these animals by affinity chromatography also possessed potent neutralization activity for several primary HIV-1 isolates. These results indicate that the V1/V2 domain of HIV-1 gp120 contains conserved epitopes that mediate potent neutralization of primary viruses, and suggest that subunit vaccines that efficiently induce such antibodies may provide protective humoral immunity against clinically relevant HIV-1 isolates.     

Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.     

Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys

Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS(p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitro gamma-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or gamma-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitro gamma-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.     

Role of individual T-cell epitopes of Theiler's virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Three major T-cell epitopes have previously been identified within the VP1 (VP1233-250), VP2 (VP274-86), and VP3 (VP324-37) capsid proteins in virus-infected SJL/J mice. These epitopes appear to account for the majority ( approximately 90%) of major histocompatibility complex class II-restricted T-cell responses to TMEV. Interestingly, the effect of immunization with synthetic peptides bearing the predominant T-cell epitopes on the course of TMEV-induced demyelination indicates that T cells reactive to the VP1 and VP2 epitopes, but not VP3, accelerate the pathogenesis of demyelination. The predominant pathogenic role of the T cells is verified by similar immunization with the fusion proteins containing the entire individual capsid proteins. The order of appearance and level of T cells specific for the individual epitopes during the course of demyelination are similar to each other. However, cytokine profiles of T cells from virus-infected mice indicate that T cells specific for the VP1 (and perhaps the VP2) epitope are Th1, whereas T cells reactive to VP3 are primarily Th2. These results suggest that Th1-type cells specific for VP1 and VP2 are involved in the pathogenesis of viral demyelination induced by TMEV. Thus, a predominance of Th1-inducing viral epitopes is likely critical for the pathogenesis of demyelination.     

In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

Cultured blood dendritic cells retain HIV-1 antigen-presenting capacity for memory CTL during progressive HIV-1 infection

Dendritic cells (DC) are potent APC that may be involved in the pathogenesis of HIV-1 infection. We studied the APC function of DC from HIV-1-infected subjects that were derived from monocyte-depleted PBMC by culture in human IL-4 and human granulocyte-macrophage CSF. The cultured cells from the HIV-1-infected subjects had similar morphology and phenotype of mature DC (CD80 = 41 +/- 8%, CD86 = 77 +/- 5%, CD40 = 87 +/- 6%, CD1a = 1 +/- 1%) to DC cultured from seronegative subjects. The yield of these DC was lower than from HIV-1-seronegative subjects (4 +/- 0% vs 11 +/- 2%, p < 0.01), and the lower DC yields correlated with lower numbers of blood CD4+ T cells (r = 0.60, p < 0.01) and higher plasma viral load (r = -0.49, p < 0.01). DC from HIV-1-infected subjects were infected with recombinant vaccinia virus vectors expressing Gag, Pol, and Env and were able to stimulate equal or higher levels of MHC class I-restricted, anti-HIV-1 memory CTL (CTLm) than were similarly treated, autologous B lymphocyte cell lines. DC pulsed with peptides representing HIV-1 CTL epitopes stimulated higher levels of anti-HIV-1 CTLm responses than did DC infected with the vaccinia virus-HIV-1 constructs. Allogeneic, MHC class I-matched DC also stimulated anti-HIV-1 CTLm activity in cells from HIV-1-infected subjects. DC from early and late stages of HIV-1 infection had a similar ability to activate CTLm specific for targets expressing either HIV-1 genes via vaccinia virus vectors or HIV-1 immunodominant synthetic peptides. However, DC from either early or late stages of HIV-1 infection could not overcome the defect in anti-HIV-1 CTLm response in advanced infection.