No abstract

Vaccinia virus is a large enveloped poxvirus with more than 200 genes in its genome. Although many poxvirus genomes have been sequenced, knowledge of the host and viral protein components of the virions remains incomplete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and host proteins in purified vaccinia intracellular mature virions (IMV). Analysis of the proteins in the IMV showed that it contains 75 viral proteins, including structural proteins, enzymes, transcription factors, and predicted viral proteins not known to be expressed or present in the IMV. We also determined the relative abundances of the individual protein components in the IMV. Finally, 23 IMV-associated host proteins were also identified. This study provides the first comprehensive structural analysis of the infectious vaccinia virus IMV.

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV bindingDengue virus (DV) is an arthropod-borne human pathogen that causes a serious public health threat in tropical and subtropical regions of the world (58). The World Health Organization reports that there are approximately 500,000 cases of dengue fever per year and that the infection rate is approximately 50 million per year (see reference 15 and literature cited therein). DV has four serotypes (DEN-1 to DEN-4) that cause diseases ranging from mild dengue fever to severe symptoms such as dengue hemorrhagic fever and dengue shock syndrome (17,25,27,70).The dengue viral genome is a single-stranded, positivestrand RNA with genome organization similar to those of other flaviviruses (47). DV infects a broad range of mammalian cell lines from several species in vitro but is transmitted to humans in vivo by mosquito vectors such as Aedes aegypti and Aedes albopictus (7). Primary human cells such as peripheral blood leukocytes, blood monocytes/macrophages, dendritic cells, and B lymphocytes could also be infected by DV (9,12,13,28,32,33,46,53,55,75,76,82).Previous studies indicate that cell surface heparan sulfates (HS) are involved in attachment of DV to mammalian cells including Vero, CHO, and human hepatoma cells (11,23,31,35,44,54). HS are repeating disaccharides composed of uronic acid or L-iduronic acid and a derivative of glucosamine that is variably O-sulfated (21). Extensive sulfate modification causes cell surface HS to be highly negatively charged. The biological roles of HS are quite diverse, including cell attachment and migration, compressive resilience of cartilage, control of fibrinogenesis, cell signaling, and virus infection (3). Many pathogenic microorganisms, including viruses, gram-positive and gram-negative bacteria, and parasites, attach to HS during entry into host cells (14,69,74). Since HS is ubiquitously expressed on many cell types and is commonly used by other pathogens to gain access into cells, an additional coreceptor has been postulated to explain the limited cell tropism of DV. This coreceptor may be related to a trypsin-sensitive protein or protein complex that was shown to play a role in virion binding to mammalian cells (18,52). Several candidate coreceptor proteins for DV have been suggested for many mammalian cell lines. These proteins are between 20 to 40 kDa and 60 to 90 kDa in size and bind to dengue virions in vitro (5,31,56,59,66). Recently, DC-SIGN, a dendritic cell surface lectin, was shown to mediate DV infection to primary dendritic cells (61,81). Nevertheless, the molecular mechanism by which DV enters these cells remains poorly characterized.Besides mammalian cells, mosquito cell lines that express cell surface proteins capable of binding to DV were also reported. In C6/36 cells, 40-to 45-kDa cell surface proteins were identified to bind 83). Additional candidate coreceptors on C6/36 cells w...

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.