Vesicular Stomatitis Virus Matrix Protein Mutations That Affect Association with Host Membranes and Viral Nucleocapsids

Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication.Most viruses that have a membrane or envelope as part of their structure acquire their envelopes by budding from the plasma membrane of the host cell. For budding to occur, the nucleoprotein core of the virus (nucleocapsid) must interact with the cytoplasmic surface of the host membrane. For many viruses this interaction is mediated by a matrix (M)² protein that binds to both the nucleocapsid and the host membrane (1, 2). Despite the similarity in the functions of viral M proteins, there is little structural or sequence similarity among the M proteins of different virus families (3). Thus, understanding the relationship of structure to function must be undertaken for individual M proteins before the general principles involved in virus budding can be understood. The goal of the experiments described here was to determine sequences in the M protein of vesicular stomatitis virus (VSV) involved in binding to membranes and nucleocapsids.VSV is the prototype member of the Rhabdoviridae family and has been widely studied to determine mechanisms involved in virus budding (2). The core of the virus contains an ∼11-kilobase negative-stranded RNA genome covered by 1300 copies of a single nucleocapsid protein (4). The nucleocapsid also contains lesser amounts of two proteins, P and L, which constitute the viral RNA-dependent RNA polymerase. The envelope contains a single species of transmembrane glycoprotein (G protein) that mediates virus attachment and entry into host cells. The virion contains ∼2000 copies of the M protein (4), which binds the nucleocapsid to the envelope and condenses the nucleocapsid into a tightly coiled helical nucleocapsid-M protein (NCM) complex that gives the virion its bullet-like shape (5-8). In cells infected with VSV and in transfected cells that express M protein in the absence of other VSV components, M protein is present both in a soluble form and bound to the cytoplasmic surface of the host plasma membrane (9-18). Mutagenesis studies, affinity labeling, and membrane reconstitution experiments have suggested that a combination of hydrophobic and ionic interactions mediate M protein binding to membranes by binding acidic phospholipids on the inner surface of the host plasma membrane (for review, see Ref. 19). Binding of M protein to nucleocapsids is less well understood than its binding to membranes. Most of the M protein in isolated NCM complexes is bound in a rapidly reversible equilibrium (20). However, M protein does not bind to nucleocapsids from which all of the M protein has been dissociated or to intracellular nucleocapsids that have never been assembled with M protein (11, 20). This suggests that binding of M protein to nucleocapsids in infected cells must be initiated in a separate step, after which most of the M protein is recruited into the NCM complex through the reversible binding step.M protein does not have separately folded domains that mediate binding to membranes versus nucleocapsids. The 229-amino acid (aa) M protein contains a positively charged N terminus (aa 1-50) that is highly exposed to proteolysis. The remainder of M protein (aa 51-229) is compactly folded to form a protease-resistant core (16, 21-23). The ability to obtain crystals of M protein required proteolytic removal of both the N-terminal sequence (aa 1-47) and a hydrophobic sequence (aa 121-124) to prevent M protein self-association (21, 22); however, the resulting structure showed a single-domain fold for the crystallized portion of M. In the present study we focused on two regions of the M protein structure that had been suggested to be involved in binding to either membranes or nucleocapsids; 1) previous data had implicated the N-terminal sequence in binding to both nucleocapsids and membranes (9, 10, 16, 22-25) and 2) deletion mutagenesis studies had implicated an additional region from aa 75-106 in membrane binding (16).In the experiments described here, M protein sequence substitutions were made using a scanning approach in the N-terminal sequence, and substitutions were based on the crystal structure in the 75-106-aa region. These mutants were used to determine the specific amino acids involved in these interactions. The results confirm that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities are differentially affected by individual mutations. Mutations in the 75-106-aa region affected incorporation into NCM complexes but had only minor effects on association with membranes. Furthermore, the ability of mutant M proteins to function in the context of virus infection suggested that a new activity of M protein that is separate from its ability to associate with membranes or NCM complexes is critical for virus assembly.     

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.     

Adelphocorisella Miyamoto and Yasunaga newly recorded from Australia, with the description of a new species (Heteroptera: Miridae: Mirinae)

Adelphocorisella australis sp. n. is described from north Queensland. This, the first representative of the genus known from Australia, is compared with the two previously described species, both from Japan.     

Survival of Trichomonas gallinae in white-winged dove carcasses

Survival of Trichomonas gallinae was examined in white-winged dove (Zenaida asiatica) carcasses to assess whether birds that have been dead up to 8 hr can be sampled reliably for this protozoan. Carcasses of 100 T. gallinae-positive white-winged doves were separated into four groups of 25 birds, representing 2, 4, 6, and 8 hr post mortem sampling intervals and placed into an environmental chamber maintained at 27 C and 75% relative humidity. Live T. gallinae were isolated in 96, 100, 100, and 92% of the carcasses at each of the respective post mortem intervals. The experiment was repeated with another 100 carcasses of T. gallinae-positive white-winged doves placed in the environmental chamber, this time maintained at 27 C and 40% relative humidity. Live T. gallinae occurred in 96, 100, 96, and 100% of the carcasses at each of the respective post mortem intervals. Across both trials, the overall ability to detect positive birds from sampling carcasses up to 8 hrs post mortem was 97%. An a posteriori experiment was conducted in which 23 and 18 carcasses from the second trial were maintained in the environmental chamber at 27 C and 40% relative humidity and resampled at 24 and 48 hr post mortem, respectively. Live trichomonads were isolated from 91 and 44% of the carcasses at 24 and 48 hr, respectively. Results suggest live T. gallinae can be obtained from dove carcasses reliably up to 8 hr and possibly up to 24 hr after host death. The ability for T. gallinae to survive within this time interval can aid wildlife personnel in monitoring this protozoan at hunter check stations or obtaining samples from recently killed birds.     

Oncolytic vesicular stomatitis virus induces apoptosis via signaling through PKR, Fas, and Daxx

Matrix (M) protein mutants of vesicular stomatitis virus (VSV) are promising oncolytic agents for cancer therapy. Previous research has implicated Fas and PKR in apoptosis induced by other viruses. Here, we show that dominant-negative mutants of Fas and PKR inhibit M protein mutant virus-induced apoptosis. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated apoptosis. However, the expression of dominant-negative FADD had little effect on the induction of apoptosis by M protein mutant VSV. Instead, virus-induced apoptosis was inhibited by the expression of a dominant-negative mutant of the adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during M protein mutant VSV infection and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.     

Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes

We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.