Constraints on HIV-1 diversity from protein structure

The high rate of HIV-1 evolution contributes to immune escape, enables the virus to escape drug therapy, and may underlie the difficulty of producing an effective vaccine. Identifying constraints on HIV evolution is therefore of prime importance. To investigate this problem, we examined the relationships between sequence diversity, selection, and protein structure. We found that while there was an increase in sequence diversity over time, this variation had a tendency to be limited to specific structural regions. When individual sites were analyzed, there was, in contrast, substantial and widespread evolutionary constraint over gag and env. This constraint was present even in the highly variable envelope proteins. The evolutionary significance of an individual site is indicated by the change in selection pressure along the time course: increasing entropy indicates that the site is evolving predominantly in a more "clock"-like manner, low entropy values with no increase indicate a high degree of constraint, and high entropy values indicate a lack of constraint. Few sites display high degrees of turnover. Mapping these sites onto the three-dimensional protein structure, we found a significant difference between evolutionary rates for regions buried in the core of the protein and those on the surface. This constraint did not change over the time period analyzed and was not subtype dependent, as similar results were found for subtypes B and C. This link between sequence and structure not only demonstrates the limits of recent HIV-1 evolution but also highlights the origins of evolutionary constraint on viral change.     

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.     

Crystal Structures of Inhibitor Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.     

Complete Genome Sequence of Staphylococcus lugdunensis Strain HKU09-01

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci and commonly found as part of the human skin flora. It is a significant cause of catheter-related bacteremia and also causes serious infections like native valve endocarditis in previously healthy individuals. We report the complete genome sequence of this medically important bacterium.Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci (CoNS) commonly colonizing the human skin and mucosal membranes. While the genus Staphylococcus contains 48 named species currently, only a few species, notably S. aureus, are coagulase positive. Thus, the phenotypic characteristic is routinely tested in the medical microbiological laboratory for rapid differentiation of the highly pathogenic S. aureus from the other staphylococci. Among the CoNS, only a few species are known to cause human disease, usually in the form of opportunistic infections only (6). However, S. lugdunensis is an important exception (3). Besides causing catheter-related bacteremia similar to other CoNS, it causes a variety of severe nosocomial and community-acquired infections, including native valve endocarditis, a devastating and potentially fatal disease that can affect previously healthy individuals. Another unusual feature are the susceptibilities of S. lugdunensis isolates to multiple antimicrobial agents even when the incidence of multiple-drug-resistant CoNS and S. aureus occurrences are increasing in both hospital and community settings (4, 5).The genome sequence of S. lugdunensis strain HKU09-01 was determined by high-throughput sequencing performed on a GS FLX system (Roche Diagnostics, Basel, Switzerland), with approximately 45-fold coverage of the genome. This clinical strain was previously isolated from the culture of pus from a skin swab. Genome assembly was performed using the Newbler assembler, resulting in 30 large contigs (>500 bp in size). The contigs were then ordered and oriented into one scaffold using OSLay (11). The genome-finishing strategy for S. lugdunensis was similar to that employed for our previously sequenced Laribacter hongkongensis genome (12). Briefly, gap closures were performed by genomic PCR followed by DNA sequencing of amplification products on an ABI 3130xl sequencer (Applied Biosystems, CA). The finished sequence was validated by genome macrorestriction analysis using multiple rare-cutting enzymes and visualization by pulsed-field gel electrophoresis. Protein coding regions were predicted with Glimmer3 (2), and automatic genome annotation was performed on the RAST server (1). Additionally, annotation of tRNA and transfer-messenger RNA (tmRNA) genes was performed using tRNAScan-SE (10) and ARAGORN (9). Identification of rRNA genes was performed using RNAmmer (8).The genome of S. lugdunensis strain HKU09-01 consists of a circular 2,658,366-bp chromosome with G+C content of 33.87%, similar to those of other staphylococci. No plasmids are present in the sequenced strain. The genome contains 61 tRNA genes for all amino acids and 2,489 predicted protein-coding genes. Eight putative genomic islands were identified, and one actually consists of a pair of duplicated 32-kb genomic regions. Similar to Staphylococcus saprophyticus (7), but different from the other staphylococci, the genome contains 6 rRNA operons, one of them having the unusual organization 5S-16S-23S-5S.With the availability of the present genome sequence, S. lugdunensis now joins other staphylococcal species with human pathogenic potential, like S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus, to have at least one reference genome available. Further in-depth analysis will be necessary to fully elucidate the genomic differences that may explain the variation in virulence of the staphylococcal species.     

Negative Feedback Control of Osteoclast Formation through Ubiquitin-mediated Down-regulation of NFATc1

The regulation of NFATc1 expression is important for osteoclast differentiation and function. Herein, we demonstrate that macrophage-colony-stimulating factor induces NFATc1 degradation via Cbl proteins in a Src kinase-dependent manner. NFATc1 proteins are ubiquitinated and rapidly degraded during late stage osteoclastogenesis, and this degradation is mediated by Cbl-b and c-Cbl ubiquitin ligases in a Src-dependent manner. In addition, NFATc1 interacts endogenously with c-Src, c-Cbl, and Cbl-b in osteoclasts. Overexpression of c-Src induces down-regulation of NFATc1, and depletion of Cbl proteins blocks NFATc1 degradation during late stage osteoclastogenesis. Taken together, our data provide a negative regulatory mechanism by which macrophage-colony-stimulating factor activates Src family kinases and Cbl proteins, and subsequently, induces NFATc1 degradation during osteoclast differentiation.