辛德毕斯病毒装配及其6K蛋白与中间纤维的关系

用温和的选择性抽提方法与整装细胞电镜技术,DGD包埋-去包埋剂电镜技术相结合,对辛德毕斯病毒的装配与宿主细胞中间纤维的关系进行了探讨。电镜观察清晰地显示了病毒“装配中心”被中间纤维所网络,病毒的装配过程显然是以中间纤维为支架;正在装配的核壳体与已装配的核壳体紧密结合在中间纤维丝上。根据电镜照片分析,核壳体可能是沿中间纤维由装配中心向外扩散。应用人工合成6K蛋白所得抗体进行胶体金标记,证明辛德毕斯病毒非结构性6K蛋白也与中间纤维紧密结合。     

Analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope

We isolated mutants whose vaccinia hemagglutinin (HA) accumulates on nuclear envelopes and the rough endoplasmic reticulum. Mutant HA must be blocked at a pre-Golgi step because it has high-mannose-type carbohydrates but no fucose. Neither N- nor O-glycosidically linked carbohydrates are involved in the transport defect of the mutant HA, because tunicamycin, an inhibitor of N-type glycosylation, has no effect, and O-type glycosylation takes place in the Golgi organelle. The unglycosylated form of the mutant HA synthesized in the presence of tunicamycin is 3000 daltons larger than the wild type. This higher molecular weight is related to the transport defect. HAs translated in vitro also show this difference, evidence that it reflects mutation in the HA structural gene. Portions of HAs that project into the cytoplasm seem to account for this weight difference. Thus the cytoplasmic tail of glycoprotein has an important function in transport out of the rough endoplasmic reticulum.     

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCP's activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCP's complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.     

Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.     

Capturing the natural diversity of the human antibody response against vaccinia virus

The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses.     

Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus.

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.     

Positive selection on the H3 hemagglutinin gene of human influenza virus A.

The hemagglutinin (HA) gene of influenza viruses encodes the major surface antigen against which neutralizing antibodies are produced during infection or vaccination. We examined temporal variation in the HA1 domain of HA genes of human influenza A (H3N2) viruses in order to identify positively selected codons. Positive selection is defined for our purposes as a significant excess of nonsilent over silent nucleotide substitutions. If past mutations at positively selected codons conferred a selective advantage on the virus, then additional changes at these positions may predict which emerging strains will predominate and cause epidemics. We previously reported that a 38% excess of mutations occurred on the tip or terminal branches of the phylogenetic tree of 254 HA genes of influenza A (H3N2) viruses. Possible explanations for this excess include processes other than viral evolution during replication in human hosts. Of particular concern are mutations that occur during adaptation of viruses for growth in embryonated chicken eggs in the laboratory. Because the present study includes 357 HA sequences (a 40% increase), we were able to separately analyze those mutations assigned to internal branches. This allowed us to determine whether mutations on terminal and internal branches exhibit different patterns of selection at the level of individual codons. Additional improvements over our previous analysis include correction for a skew in the distribution of amino acid replacements across codons and analysis of a population of phylogenetic trees rather than a single tree. The latter improvement allowed us to ascertain whether minor variation in tree structure had a significant effect on our estimate of the codons under positive selection. This method also estimates that 75.6% of the nonsilent mutations are deleterious and have been removed by selection prior to sampling. Using the larger data set and the modified methods, we confirmed a large (40%) excess of changes on the terminal branches. We also found an excess of changes on branches leading to egg-grown isolates. Furthermore, 9 of the 18 amino acid codons, identified as being under positive selection to change when we used only mutations assigned to internal branches, were not under positive selection on the terminal branches. Thus, although there is overlap between the selected codons on terminal and internal branches, the codons under positive selection on the terminal branches differ from those on the internal branches. We also observed that there is an excess of positively selected codons associated with the receptor-binding site and with the antibody-combining sites. This association may explain why the positively selected codons are restricted in their distribution along the sequence. Our results suggest that future studies of positive selection should focus on changes assigned to the internal branches, as certain of these changes may have predictive value for identifying future successful epidemic variants.     

Evidence against an essential role of COPII-mediated cargo transport to the endoplasmic reticulum-Golgi intermediate compartment in the formation of the primary membrane of vaccinia virus

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1(H79G), a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1(H79G) by transfection or by a novel recombinant vaccinia virus with an inducible Sar1(H79G) gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1(H79G) overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.     

Interaction of vaccinia virus with the actin cytoskeleton

Vaccinia virus infection results in large rearrangements of the host actin cytoskeleton including the formation of actin tails that are strikingly similar to those seen inListeria, Shigella andRickettsia infections. Using actin polymerization as the driving force the intracellular enveloped form of the vaccinia virus (IEV) is propelled on the tip of actin tails at a speed of 2.8 μm/min, both intra- and intercellularly. The similarities between the actin-based motility of the vaccinia virus,Listeria, Shigella andRickettsia suggest that intracellular pathogens have developed a common strategy to exploit the actin cytoskeleton of the host to facilitate their intercellular spread. This review focuses on our current understanding of the interactions between the vaccinia virus,and the actin cytoskeleton. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

Metabolic efficiency in courtship favors males with intermediate mass in the Australian redback spider,Latrodectus hasselti

Recent studies have suggested that metabolic efficiency may be an important factor in male mating success when females require vigorous and/or prolonged courtship. In capital breeding animals in which a male’s resource pool is fixed at adulthood the relationship between energy expenditure and courtship performance may be especially important, as males are expected to utilize their finite resources efficiently when soliciting mates. Males may benefit from being efficient, i.e., achieving a sufficiently high level of courtship signaling at low energetic cost, if it enables them to acquire mates before their limited energy reserves are depleted. We investigated the relationship between metabolic efficiency and courtship vibrational signaling in the Australian redback spider, Latrodectus hasselti, a semelparous capital breeder where males invest heavily in courtship to secure a mating. We assessed metabolic rate in a sample of males and measured two courtship components (duty cycle and amplitude) that reflected the energy content of web-borne vibrations. We then calculated two indices of metabolic efficiency for these courtship properties. There was a quadratic relationship between mass and duty cycle such that the highest duty cycle signals were performed by males having intermediate mass. Furthermore, intermediate-mass males were also the most metabolically efficient. Prolonged courtship is necessary in L. hasselti for successful mating, and the results of this study suggest that intermediate-mass males are superior courters because they utilize their finite resource pool most efficiently to produce high energy vibrational signals.