Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells

T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.     

A Conserved Domain in the Coronavirus Membrane Protein Tail Is Important for Virus Assembly

Coronavirus membrane (M) proteins play key roles in virus assembly, through M-M, M-spike (S), and M-nucleocapsid (N) protein interactions. The M carboxy-terminal endodomain contains a conserved domain (CD) following the third transmembrane (TM) domain. The importance of the CD (SWWSFNPETNNL) in mouse hepatitis virus was investigated with a panel of mutant proteins, using genetic analysis and transient-expression assays. A charge reversal for negatively charged E₁₂₁ was not tolerated. Lysine (K) and arginine (R) substitutions were replaced in recovered viruses by neutrally charged glutamine (Q) and leucine (L), respectively, after only one passage. E₁₂₁Q and E₁₂₁L M proteins were capable of forming virus-like particles (VLPs) when coexpressed with E, whereas E₁₂₁R and E₁₂₁K proteins were not. Alanine substitutions for the first four or the last four residues resulted in viruses with significantly crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. All recovered viruses with alanine substitutions in place of SWWS residues had second-site, partially compensating, changes in the first TM of M. Alanine substitution for proline had little impact on the virus. N protein coexpression with some M mutants increased VLP production. The results overall suggest that the CD is important for formation of the viral envelope by helping mediate fundamental M-M interactions and that the presence of the N protein may help stabilize M complexes during virus assembly.Coronaviruses are widespread, medically important respiratory and enteric pathogens of humans and a wide range of animals. New human coronaviruses (HCoV), including severe acute respiratory syndrome CoV (SARS-CoV), HCoV-NL63, and HCoV-HKU1, were recently identified (40, 47). The potential for emergence of other new viruses and the zoonotic nature of some coronaviruses strongly warrants understanding old and new viruses. Understanding vital interactions that take place during virus assembly and conserved domains (CDs) that mediate these interactions can provide insight toward identification of targets for development of antiviral therapeutics and vaccines.Coronaviruses are enveloped positive-stranded RNA viruses that belong to the Coronaviridae family in the Nidovirales order. The virion envelope contains at least three structural proteins, the membrane (M), spike (S), and envelope (E) proteins. The genomic RNA is encapsidated by the N phosphoprotein to form a helical nucleocapsid. The S glycoprotein is the viral attachment protein that facilitates infection through fusion of viral and cellular membranes and is the major target of neutralizing antibodies (13). The M glycoprotein is the most abundant component of the viral envelope and plays required, key roles in virus assembly (9, 20, 31, 33, 41). The E protein is a minor component of the viral envelope that plays an important, not clearly defined, role(s) during virus assembly and release (2, 5, 41).Coronavirus M proteins are divergent in their amino acid content, but all share the same overall basic structural characteristics. The proteins have three TM domains, flanked by a short amino-terminal glycosylated domain and a long carboxy-terminal tail located outside and inside the virion, respectively (14) (Fig. ​(Fig.11 A). M localizes in the Golgi region when expressed alone (20, 22). M molecules interact with each other and also with the spike and nucleocapsid during virus assembly (8-10, 23, 31, 33). M-M interactions constitute the overall scaffold for the viral envelope. The S protein and a small number of E molecules are interspersed in the M protein lattice in mature virions. Previous studies from a number of labs implicated multiple M domains and residues as being important for coronavirus assembly (6, 8, 9, 17, 43). Coronaviruses assemble and bud at intracellular membranes in the region of the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) (22, 39). Coexpression of only the M and the E proteins is sufficient for virus-like particle (VLP) assembly for most coronaviruses (2, 41).Open in a separate windowFIG. 1.M protein conserved domain and mutants. (A) A linear schematic of the M protein illustrating the relative positions of the three TM domains (black boxes) and the position of the CD in the tail. (B) Alignment of CDs from representative coronaviruses. Full-length amino acid sequences from transmissible gastroenteritis virus (TGEV), feline coronavirus (FeCoV), human coronavirus 229E, human coronavirus NL63, mouse hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43, porcine hemagglutinating encephalomyelitis virus (HEV), human coronavirus HKU1, SARS-CoV, infectious bronchitis virus (IBV), and turkey coronavirus (TCoV) were aligned by using CLUSTAL W (25). (C) Mutations introduced into the MHV CD, with + and − symbols used to indicate VLP production and virus recovery for each mutant.The long intravirion (cytoplasmic) tail of M consists of an amphipathic domain following the third TM and a short hydrophilic region at the carboxyl end of the tail (Fig. ​(Fig.1A).1A). The amphipathic domain appears to be closely associated with the membrane (34). At the amino terminus of the amphipathic domain, there is a highly conserved 12-amino-acid domain (SWWSFNPETNNL), consisting of residues 114 to 125 in the mouse hepatitis virus (MHV) A59 M protein (Fig. ​(Fig.1B)1B) (19). These residues are almost identically conserved across the entire Coronaviridae family. Because of the crucial role that M plays in virus assembly and the high conservation of this domain, we hypothesized that it is functionally important for virus assembly. To test this, a series of changes were introduced in the CD. The functional impact of the changes was studied in the context of the virus by genetic analysis and the ability of the mutant M proteins to participate in VLP assembly. The results show that the CD is functionally important for M protein to participate in virus assembly. The domain may help mediate important lateral interactions between M molecules. The results suggest that the N protein helps stabilize M complexes during virus assembly.     

Hallmarks of molecular action of microtubule stabilizing agents: effects of epothilone B, ixabepilone, peloruside A, and laulimalide on microtubule conformation

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αβ-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.     

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.     

Testing Refrigeration Trucks for the Emergency Evacuation of Companion Animals

The purpose of this study was to quantify the changes in oxygen (O₂) and carbon dioxide (CO₂) in sealed refrigerator trucks scheduled to be used for transporting companion animals (dogs and cats) during an emergency evacuation. A total of 122 nonhuman animals (total weight = 1,248 kg) housed in individual crates were loaded into a 16-m refrigeration truck. Once they were loaded, the doors were closed and the percentages of O₂ and CO₂ were measured every 5 min by O₂ and CO₂ analyzers, and they were used to quantify the changes in gas pressure in the sealed truck. CO₂ had a much higher-than-predicted increase, and O₂ had a higher-than-predicted decrease. These 2 pressures in combination with the functionality of the respiratory system will limit the animal's ability to load O₂, and over time, they will initiate asphyxia or suffocation. Over time, the partial pressure of oxygen (P_O2) in the sealed truck will decrease, causing hypoxia, and the partial pressure of carbon dioxide (P_CO2) will increase, causing hypercapnia.     

Hereditary multiple exostoses (EXT): mutational studies of familial EXT1 cases and EXT-associated malignancies.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma.     

Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET

Biochemical and functional evidence suggest that the calcitonin receptor-like receptor (CRLR) interacts with receptor activity-modifying protein-1 (RAMP1) to generate a calcitonin gene-related peptide (CGRP) receptor. Using bioluminescence resonance energy transfer (BRET), we investigated the oligomeric assembly of the CRLR-RAMP1 signaling complex in living cells. As for their wild-type counterparts, fusion proteins linking CRLR and RAMP1 to the energy donor Renilla luciferase (Rluc) and energy acceptor green fluorescent protein (GFP) reach the cell surface only upon coexpression of CRLR and RAMP1. Radioligand binding and cAMP production assays also confirmed that the fusion proteins retained normal functional properties. BRET titration experiments revealed that CRLR and RAMP1 associate selectively to form heterodimers. This association was preserved for a mutated RAMP1 that cannot reach the cell surface, even in the presence of CRLR, indicating that the deficient targeting resulted from the altered conformation of the complex rather than a lack of heterodimerization. BRET analysis also showed that, in addition to associate with one another, both CRLR and RAMP1 can form homodimers. The homodimerization of the coreceptor was further confirmed by the ability of RAMP1 to prevent cell surface targeting of a truncated RAMP1 that normally exhibits receptor-independent plasma membrane delivery. Although the role of such dimerization remains unknown, BRET experiments clearly demonstrated that CRLR can engage signaling partners, such as G proteins and beta-arrestin, following CGRP stimulation, only in the presence of RAMP1. In addition to shed new light on the CRLR-RAMP1 signaling complex, the BRET assays developed herein offer new biosensors for probing CGRP receptor activity.     

Altered protein expression at early-stage rat hepatic neoplasia

Protein expression patterns were analyzed in a rat model of hepatic neoplasia to detect changes reflecting biological mechanism or potential therapeutic targets. The rat resistant hepatocyte model of carcinogenesis was studied, with a focus on the earliest preneoplastic lesion visible in the liver, the preneoplastic hyperplastic nodule. Expression differences were shown by two-dimensional polyacrylamide gel electrophoresis and image analysis. Polypeptide masses were measured by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and their sequences were obtained by tandem mass spectrometry. Alterations in expression of cytoskeletal and functional proteins were demonstrated, consistent with biological changes known to occur in the preneoplastic cells. Of particular interest was the differential expression of a serine protease inhibitor (serpin) with a role implicated in angiogenesis. Serpin, implicated in the inhibition of angiogenesis, is present in normal liver but has greatly reduced expression at the preneoplastic stage of liver cancer development. Immunofluorescence microscopy with antibodies to this serpin, kallistatin, supports the proteomic identification. Immunofluorescence microscopy with antibodies to the blood vessel marker von Willebrand factor provides evidence for neovascularization in the liver containing multiple preneoplastic nodules. These observations suggest that at an early stage of liver carcinogenesis reduction or loss of angiogenesis inhibitors may contribute to initiation of neoangiogenesis. A number of other identified proteins known to be associated with hepatomas are also present at early-stage neoplasia.     

Mouse hepatitis coronavirus A59 nucleocapsid protein is a type I interferon antagonist

The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2',5'-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VVDeltaE3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VVDeltaE3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.