Glycosylation affects both the three-dimensional structure and antibody binding properties of the HIV-1IIIB GP120 peptide RP135.

We have prepared glycosylated analogues of the principal neutralizing determinant of gp120 and studied their conformations by NMR and circular dichroism spectroscopies. The 24-residue peptide from the HIV-1IIIB isolate (residues 308-331) designated RP135, which contains the immunodominant tip of the V3 loop, was glycosylated with both N- and O-linked sugars. The structures of two glycopeptides, one with an N-linked beta-glucosamine (RP135NG) and the other with two O-linked alpha-galactosamine units (RP135digal), were studied by NMR and circular dichroism spectroscopies. Molecular dynamics calculations based on the NMR data obtained in water solutions were performed to explore the conformational substates sampled by the glycopeptides. The data showed that covalently linking a carbohydrate to the peptide has a major effect on the local conformation and imparts additional minor changes at more distant sites of partially defined secondary structure. In particular, the transient beta-type turn comprised of the -Gly-Pro-Gly-Arg- segment at the "tip" of the V3 loop is more highly populated in RP135digal that in the native peptide and N-linked analogue. Binding data for the glycopeptides with 0.5beta, a monoclonal antibody mapped to the RP135 sequence, revealed a significant enhancement in binding for RP135digal as compared with the native peptide, whereas binding was reduced for the N-linked glycopeptide. These data show that glycosylation of V3 loop peptides can affect their conformations as well as their interactions with antibodies. The design of more ordered and biologically relevant conformations of immunogenic regions from gp120 may aid in the design of more effective immunogens for HIV-1 vaccine development.

Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope.

Immunodominant epitopes are known to suppress a primary immune response to other antigenic determinants by a number of mechanisms. Many pathogens have used this strategy to subvert the immune response and may be a mechanism responsible for limited vaccine efficacies. HIV-1 vaccine efficacy appears to be complicated similarly by a limited, immunodominant, isolate-restricted immune response generally directed toward determinants in the third variable domain (V3) of the major envelope glycoprotein, gp120. To overcome this problem, we have investigated an approach based on masking the V3 domain through addition of N-linked carbohydrate and reduction in net positive charge. N-linked modified gp120s were expressed by recombinant vaccinia virus and used to immunize guinea pigs by infection and protein boosting. This modification resulted in variable site-specific glycosylation and antigenic dampening, without loss of gp120/CD4 binding or virus neutralization. Most importantly, V3 epitope dampening shifted the dominant type-specific neutralizing Ab response away from V3 to an epitope in the first variable domain (V1) of gp120. Interestingly, in the presence of V3 dampening V1 changes from an immunodominant non-neutralizing epitope to a primary neutralizing epitope with broader neutralizing properties. In addition, Ab responses were also observed to conserved domains in C1 and C5. These results suggest that selective epitope dampening can lead to qualitative shifts in the immune response resulting in second order neutralizing responses that may prove useful in the fine manipulation of the immune response and in the development of more broadly protective vaccines and therapeutic strategies.

Preliminary in vitro growth cycle and transmission studies of HIV-1 in an autologous primary cell assay of blood-derived macrophages and peripheral blood mononuclear cells.

Recent interest focused on the dynamics of HIV-1 replication in primary monocytes/macrophages and T-lymphocytes of the immune system, as well as the standardization of virological and immunological in vitro assays with primary isolates, provided the impetus for these studies. These types of studies have never been performed as they would occur in vivo, i.e., where the envelope of the virus and cell membranes of the two cell types of the same host origin. Therefore, the biological and physicochemical properties of an uncloned, primary dual-tropic isolate HIV-1ADA during the initial lag, log, and stationary phases of viral replication were studied in an autologous donor cell assay in peripheral blood mononuclear cells (PBMC) and blood monocyte-derived macrophages (MDM). Similar total numbers (10(9) virus particles/ml) were produced by both cell types during the stationary period. On a per cell per day basis, during peak stationary periods, 0.92 x 10(3) virions/day for MDMs and 5.31 x 10(3) virions/day for PBMCs were produced. Interestingly, virus replicating from MDMs during the log-growth phase demonstrated the greatest infectious fraction which was 3 logs greater than virus replicating in PBMCs. Despite constant virus particle production in MDMs, the infectious fraction was found to fall 3 to 4 logs over a 10-day period. Due to an infectious fraction less than 1 (0.053 infectious unit/cell/24 hr), virus spread in PBMCs during the rapid log phase could only have occurred by cell-to-cell contact, whereas in MDMs with an infectious fraction of about one infectious particle (approximately 1/cell/24 hr), cell-free transmission could account for the observed results. Most of the MDMs (> 90%) became productively infected, whereas only 5-10% of the total PBMCs were found replicating virus. The period of peak stationary virus production (i.e., stationary phase) was at minimum 4 to 5 times longer in MDMs than PBMCs. Whereas the majority of p24, RT, and gp 120 found to be associated with MDM-derived virions, no increased dissociation of these components was observed in PBMC-derived virions. The virion-associated gp 120 was 3 to 4 times more stable on both PBMC- and MDM-derived virus (> 96 hr) and present at 10-25 times the concentration per virion than that observed for a T-cell-line-adapted laboratory strain of HIV-1 replicating in T-cell lines. These in vitro results suggest that important differences exist between MDMs and PBMCs with regard to the viral dynamics of infection and replication which should provide for a qualitative and quantitative basis to estimate virus replication on a per-cell basis for other known cellular targets of HIV-1. Studying the multiple biophysicochemical characteristics and viral replication dynamics as described herein provides an autologous in vitro model of additional quantifiable parameters for analysis and understanding of virus/host factor(s) and/or antivirals which influence them.

Neutralization of HIV-1: a paradox of humoral proportions.

The production of immunoglobulin capable of neutralizing the infectivity of a virus represents one of the most remarkable molecular accomplishments of the host's available immune defenses. It should be no surprise that a virus that has existed in the parenchyma of the immune system has evolved as an equally dynamic molecule (i.e., viral envelope) for survival. Neutralizing immunoglobulin (Ig) can best serve the host under conditions where the invading pathogen requires a well-defined cell-free state for establishing an infection or transmission. Evidence for a controlling and therefore protective role of neutralizing Ig against lentiviruses has been defined in natural and experimental infections with equine infectious anemia virus of ungulate members in the family equidae. Rapid replication of the virus immediately after infection and its release in a cell-free state leads to the production of neutralizing Ig and subsequent control of the primary viremia. A similar cause-effect relationship exists in humans between the high-titered viremia, observed shortly after HIV-1 infection, and the subsequent production of neutralizing Ig. Partially controlling this acute stage of viral replication by neutralizing Ig and thus preventing an otherwise acute form of immunosuppression or immune complex disease may be viewed paradoxically as a survival property of the virus. Immunologically mediated control, in a Darwinian sense, selects for viruses that have optimized the parameters of longevity and transmission from host to host. This paradox of neutralization in HIV-1 infection appears to be mediated by the convergence of structural and functional roles of the third variable domain (V3) of the external envelope glycoprotein. During infection or envelope-based vaccination, antibody to this cross-reactive, immuno-dominant epitope dominates the antigenic repertoire. Once this occurs, the host is less able to respond to emerging viruses containing closely related V3 structures. Thus a relatively restricted clonal-dominance of the neutralization response results. The V3 domain, apparently in concert with the rest of the molecule, provides an epitope that can tolerate and utilize its conformational flexibility to allow immune escape while maintaining its functional role in infectivity. Sixteen other putative epitopes have been described as being involved in the induction of neutralizing Ig. Currently the biologically functional role of neutralizing Ig to these other epitopes are complicated by a prior lack of knowledge and appreciation of the in vitro variables affecting their measurements.(ABSTRACT TRUNCATED AT 400 WORDS)

Deletion mapping of tumor promotion-susceptibility gene pro1 implicates an RNA polymerase III transcription unit.

The murine gene pro1 has been cloned from JB6 epidermal cell lines that are sensitive to neoplastic transformation by tumor promoters. Insensitive JB6 variants acquire susceptibility to neoplastic transformation by tumor promoters when transfected with pro1. The repetitive nature of pro1 was indicated by sequence and Southern analysis. In contrast, northern analysis of RNA from promotion-sensitive cells revealed the presence of a small pro1-hybridizing transcript. Strand-specific RNA probes implicated an RNA polymerase III (RNAPIII) coding domain in pro1 as the source of this hybridization signal. Ribonuclease protection of gel-purified pro1 RNA from JB6 variant cell lines identified a 130-nucleotide transcript. The size of this transcript is compatible with in vitro RNAPIII transcription of pro1. Deletion mapping of pro1 by exonuclease III demonstrated that the biologically active domain included the RNAPIII transcription unit. RNA probes map pro1 RNA within the activity domain. These results delineate an activity domain of 597 nucleotides and suggest that a small RNA is the product of promotion-sensitivity gene pro1.

Evidence that mouse promotion-sensitivity gene pro1 is transcribed by RNA polymerase III.

The murine gene pro1 confers susceptibility to tumor promoters upon transfection into an insensitive host cell. Nucleotide analysis over a minimally active domain of 1049 bp reveals signals expected for a gene transcribed by RNA polymerase II (RNAPII). Similar analysis of the complementary strand shows intragenic signals characteristic of genes transcribed by RNA polymerase III (RNAPIII). We have previously characterized a small, pro1-homologous transcript that is constitutively expressed at lower levels in promotion-insensitive JB6 epidermal cells as compared to promotion-sensitive and transformed clonal variants. To identify whether the pro1 RNAPII or RNAPIII transcription unit encodes the pro1-homologous RNA, RNA probes specific for each of the predicted transcripts were generated. The RNA probe specific for the pro1 RNAPIII transcription unit was found to detect the pro1-hybridizing RNA. Ligating the pro1 RNAPII 5'-flanking region to an interferon gamma reporter sequence failed to induce synthesis of the reporter protein. In addition, pro1 transcripts generated from the predicted RNAPII and RNAPIII transcription units were untranslatable in rabbit reticulocyte lysates. These data are consistent with pro1 associated tumor promotion occurring not through an RNAPII intermediate, but through an RNAPIII intermediate.

Inhibition of T7 bacteriophage replication by a colicin Ib plasmid gene.

Clones containing fragments of the colicin Ib (ColIb) plasmid inserted into pBR322 have been found that inhibit the replication of T7 bacteriophage. Cells containing the whole ColIb plasmid grow T7 normally but cannot grow T7 protein kinase-negative mutants of T7. The cloned fragments inhibit not only the T7 protein kinaseless mutants but wild-type T7 as well. However, the whole plasmid can suppress the wild-type T7 inhibition caused by the cloned inhibiting genes. These results are consistent with a model in which a ColIb gene (pic) exists which can inhibit replication of T7 phage. A second gene (rpi) can repress the function of pic provided the rpi product is phosphorylated.