Macrophages and Cell-Cell Spread of HIV-1

Macrophages have been postulated to play an important role in the pathogenesis of HIV-1 infection. Their ability to cross the blood-brain barrier and their resistance to virus-induced cytopathic effects allows them to serve as reservoirs for long-term infection. Thus, exploring the mechanisms of virus transmission from macrophages to target cells such as other macrophages or T lymphocytes is central to our understanding of HIV-1 pathogenesis and progression to AIDS, and is vital to the development of vaccines and novel antiretroviral therapies. This review provides an overview of the current understanding of cell-cell transmission in macrophages

Plasma Levels of High-Mobility Group Box 1 during Peptide Vaccination in Patients with Recurrent Ovarian Cancer

High-mobility group box 1 (HMGB1) is a nuclear protein that is known to be secreted into extracellular fluids from injured cells, activated macrophages, and tumor cells. The clinical correlation of circulating HMGB1 levels with various diseases including cancer has been reported. However, there is no information on HMGB1 levels in cancer patients treated with peptide vaccination. In the present study, we investigated the plasma levels of HMGB1 during personalized peptide vaccination in patients with recurrent ovarian cancer. Frozen plasma samples of 39 patients from previously conducted clinical trials were used in this study. HMGB1 levels were decreased after the 1st cycle of vaccination from their prevaccination levels. However, no correlation was observed between HMGB1 and overall survival (OS). The correlation between plasma HMGB1 levels and other biomarker levels was further analyzed by scatter plot, revealing that HMGB1 levels after the 1st cycle of vaccination were significantly correlated with myeloid-derived suppressor cell (MDSC) frequency after the 1st cycle of vaccination (r=0.357, p=0.032). Chi-square test showed that epitope spreading was significantly related with changes of HMGB1 (p=0.030). These results suggest that plasma HMGB1 is a possible biomarker for cancer vaccine therapy, although direct correlation with OS has not been obtained. This trial is registered with Clinical Trial Registry under trial numbers UMIN000003083 and UMIN000001482

Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania-Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum N-Acetylglucosamine-1-Phosphate Transferase

A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes

Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket

<div><p>Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for <em>in vitro</em>. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.</p> </div

PF-46396-dependent mutants are not rescued by BVM.

<p>[A] Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence (−) and presence (+) of 2 µM BVM. Positions of Pr55^Gag (PrGag), Pr41^Gag (p41), CA-SP1 and CA are indicated. Lower panels show phosphorimager-based quantification of relative virus release efficiency (VRE) and % virion CA-SP1, calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 4. [B] Virus replication kinetics in the absence and presence of BVM. The Jurkat T-cell line was transfected with WT or mutant pNL4-3 and propagated in the presence of 0, 0.1, or 1.0 µM BVM. Virus replication was monitored by RT activity, shown in cpm/µl.</p

The SP1-V7A polymorphism has a lesser effect on sensitivity to PF-46396 than to BVM.

<p>293T cells were transfected with WT pNL4-3 or pNL4-3 derivatives bearing SP1-V7A or SP1-T8A polymorphisms and treated with 1 µM BVM or PF-46396. CA-SP1 processing efficiency was examined in virions by radioimmunoprecipitation analysis. P values: **, p<0.01; dashed line, no significant difference. Error bars indicate SD; N = 3.</p

Schematic molecular model of PF-46396 and BVM binding sites.

<p>The ribbon diagrams illustrate two adjacent CA-CTD/SP1 monomers in the hexagonal complex of the immature capsid. The atomic coordinates of the CA-CTD and SP1 domains were obtained from PDB 3H4E <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Pornillos1" target="_blank">[66]</a> and PDB 1U57 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Morellet1" target="_blank">[27]</a>, respectively. Residues 220–223 that link the CA-CTD and SP1 domains of each monomer were not modeled due to a lack of experimental data. The relative orientations of the adjacent CA-CTD domains were taken from the models of Bharat et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Bharat1" target="_blank">[61]</a>. The sites of resistance mutations are labeled and color-coded for location: Green, blue and red for the MHR, CA-CTD/SP1 boundary, and individual I201 residue, respectively. Yellow indicates the sites of secondary substitutions that rescue the G156E and P157S mutants. Also shown are stick figures of the PF-46396 and BVM compounds at the heights of their respective binding sites predicted from the locations of the resistance mutations (color-code: C – brown, N - blue, O - red, H – white, and Fl – green). The rotational orientation of PF-46396 is arbitrary, but that of BVM is taken from the photoafinity data of Nguyen et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Nguyen1" target="_blank">[45]</a>. The proximity of the MHR of the left monomer to the CA-CTD/SP1 region of the right one suggests that the binding sites of the two compounds straddle adjacent monomers. This would explain the observed necessity of Gag assembly into the immature capsid structure for BVM cleavage inhibition (see text).</p

Summary of PF-46396-resistance mutations selected.

#<p>From ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Adamson5" target="_blank">[51]</a>.</p>*<p>In parentheses is the number of times the indicated mutation was selected.</p

Second-site compensatory changes for PF-46396-dependent CA mutants.

<p>Second-site compensatory changes for PF-46396-dependent CA mutants.</p