Micro Radiometric Method for Study of Acid-insoluble Materials in Monolayer Cell Cultures

A simple radiometric procedure for study of acid-insoluble products synthesized in monolayer cell cultures is described. Cell cultures were produced directly on the bottom surface of scintillation vials or on glass cover slips (8 X 30 mm). The cells were labeled and extracted; the radioactivity was determined while the cells remained affixed to the glass surface upon which they were grown. This procedure enabled rapid investigations of certain biosynthetic processes to be carried out by using many individual cell cultures. The method was applied to an investigation of (3)H-thymidine incorporation induced by vaccinia virus in a 5-bromodeoxyuridine-resistant cell line. (14)C-labeling was evaluated as an alternate procedure for cell quantitation.     

A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.     

Anti-Vaccinia Activity of γ-Thiochromanone-1-Thiosemicarbazone In Vitro and In Vivo

A derivative of thiosemicarbazone, γ-thiochromanone-l-thiosemicarbazone (SN-13), which differed from N-methylisatin-β-thiosemicarbazone (marboran) in that the carbonyl group in the C₂ position of N-methylisatin was lacking, has been found to possess an anti-vaccinia effect as determined by the pulp disc method of plaque inhibition and by inhibition of cytopathic effect in tube cultures of chick embryo cells as well as by prevention of mouse tail lesions by the vaccinia virus. In tube cultures, SN-13 was shown to be effective even when the treatment was started as late as 8 hr after virus infection, whereas no activity was observed with marboran when started from the 8th hr. SN-13 was as effective as marboran on cross treatments of vaccinia virus with the two compounds in tube cultures, either by treatment at an early or a late stage of the virus growth. Moreover, the inhibitory effect of SN-13 on vaccinia virus growth was completely reversed by actinomycin D similar to that observed with marboran in tube cultures. No additive effect of the two compounds was observed in animal tests.     

The envelope G3L protein is essential for entry of vaccinia virus into host cells

The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-beta-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L(+)) or lacking (G3L(-)) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L(+) and G3L(-) IMV bound to HeLa cells; however, G3L(-) IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.     

Vaccinia virus WR53.5/F14.5 protein is a new component of intracellular mature virus and is important for calcium-independent cell adhesion and vaccinia virus virulence in mice

The vaccinia virus WR53.5L/F14.5L gene encodes a small conserved protein that was not detected previously. However, additional proteomic analyses of different vaccinia virus isolates and strains revealed that the WR53.5 protein was incorporated into intracellular mature virus (IMV). The WR53.5 protein contains a putative N-terminal transmembrane region and a short C-terminal region. Protease digestion removed the C terminus of WR53.5 protein from IMV particles, suggesting a similar topology to that of the IMV type II transmembrane protein. We generated a recombinant vaccinia virus, vi53.5L, that expressed WR53.5 protein under isopropyl-beta-d-thiogalactopyranoside (IPTG) regulation and found that the vaccinia virus life cycle proceeded normally with or without IPTG, suggesting that WR53.5 protein is not essential for vaccinia virus growth in cell cultures. Interestingly, the C-terminal region of WR53.5 protein was exposed on the cell surface of infected cells and mediated calcium-independent cell adhesion. Finally, viruses with inactivated WR53.5L gene expression exhibited reduced virulence in mice when animals were inoculated intranasally, demonstrating that WR53.5 protein was required for virus virulence in vivo. In summary, we identified a new vaccinia IMV envelope protein, WR53.5, that mediates cell adhesion and is important for virus virulence in vivo.     

THE DEVELOPMENT OF VACCINIA VIRUS IN EARLE'S L STRAIN CELLS AS EXAMINED BY ELECTRON MICROSCOPY

A favorable system which is amenable to frequent and reproducible sampling, consisting of suspension cultures of strain L cells and vaccinia virus, was employed to study the animal virus-mammalian host cell relationship. The three principal aspects investigated concerned the adsorption and penetration of vaccinia into the host, the relationship between the sequence of virus development and the production of infectious particles, and the changes in the fine structure of the host cells. Experiments in which a very high multiplicity of infection was used revealed that vaccinia is phagocytized by L cells in less than 1 hour after being added to the culture, without any apparent loss of its outer limiting membranes. Regions of dense fibrous material, thought to be foci of presumptive virus multiplication, appear in the cytoplasm 2 hours after infection. A correlation between electron microscope studies and formation of infectious particles shows that although immature forms of the virus appear 4 hours after infection, infectious particles are produced 6 hours after infection of the culture, at the time when mature forms of vaccinia appear for the first time in thinly sectioned cells. Spread of the infection is gradual until eventually, after 24 hours, virus is being elaborated throughout the cytoplasm. Addition of vaccinia to monolayer cultures induced fusion of L cells and rapid formation of multinucleate giant forms. In both suspension and stationary cultures infected cells elaborate a variety of membranous structures not present in normal L cells. These take the form of tube-like lamellar and vesicular formations, or appear as complex reticular networks or as multi-laminar membranes within degenerating mitochondria.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

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.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates.     

The lipid raft-associated protein CD98 is required for vaccinia virus endocytosis

Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis.     

人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大,结构复杂而特殊,不可能将外源基因直接插入它的基因组,必须利用一种特殊的痘苗病毒质粒,才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,简称ＴＫ基因）,及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ,简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上,设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在,进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒,并以人体成纤维细胞作为宿主细胞,对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体,可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．     

Vaccinia Virus Replication and Cytopathic Effect in Cultures of Phytohemagglutinin-treated Human Peripheral Blood Leukocytes

Titers of vaccinia virus consistently increased in cultures of washed phytohemagglutinin-treated, peripheral blood leukocytes of a vaccinated adult. Concomitantly, a gradual rise occurred in the numbers of infected leukocytes, as determined by the infective center assay. Increase in viral titer was accompanied by cell injury, decline in cell numbers, and decreased acid production. Leukocytes not pretreated with phytohemagglutinin appeared to form infective centers after exposure to the vaccinia agent, but they did not replicate infectious virus. For viral replication, the continuous presence of phytohemagglutinin was required.     

The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus.

The mechanisms allowing vaccinia virus to spread from cell to cell are incompletely understood. The A34R gene of vaccinia virus encodes a glycoprotein that is localized in the outer membranes of extracellular virions. The small-plaque phenotype of an A34R deletion mutant was similar to that of mutants with deletions in other envelope genes that fail to produce extracellular vaccinia virions. Transmission electron microscopy, however, revealed that the A34R mutant produced numerous extracellular particles that were labeled with antibodies to other outer-envelope proteins and with protein A-colloidal gold. Fluorescence and scanning electron microscopy indicated that expression of the A34R protein was necessary for detection of vaccinia virus-induced actin tails, which provide motility to the intracellular enveloped form of vaccinia virus, and of virus-tipped specialized microvilli that project from the cell. The ability of vaccinia virus-infected cells to form syncytia after a brief exposure to a pH below 6, known as fusion from within, failed to occur in the absence of expression of the A34R protein; nevertheless, purified A34R- virions were capable of mediating low-pH-induced fusion from without. The present study provides genetic and microscopic evidence for the involvement of a specific viral protein in the formation or stability of actin-containing microvilli and for a role of these structures in cell-to-cell spread rather than in formation of extracellular virions.     

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Comparison of Transformation and T Antigen Induction in Human Cell Lines

Skin fibroblast cultures were established from eight individuals. These cell cultures, together with WI-38 cells, were examined for susceptibility to transformation by SV40 virus. Four transformation-susceptible cell lines (TS), established from patients with Down's syndrome, were found to be three to four times more susceptible to transformation than transformation-resistant cell lines (TR) from normal individuals. TR and TS cell lines were compared for their susceptibility to induction of SV40 T antigen. For dividing cells T antigen was detected in a higher percentage of TS cells than TR cells. For nondividing cells, the reverse was found; T antigen was detected in 10-fold more cells of the TR lines than in cells of the TS lines. Similar results were obtained after infection of cells with CELO virus. Titration of vaccinia virus and influenza virus A2/Scotland/49/57 indicated that TR and TS cells were equally sensitive to the former virus, but TR cells were three to five times more sensitive to influenza virus A2/Scotland/49/57 than were TS cells.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

Mechanism of Vaccinia Virus Release and Its Specific Inhibition by N1-Isonicotinoyl-N2-3-Methyl-4- Chlorobenzoylhydrazine

The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.     

Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions

Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.     

一种以我国痘苗病毒天坛株血凝素基因为选择标记的新型系列表达载体的构建

本文作者从痘苗病毒天坛株Sal I基因文库中确定并分离了痘苗病毒血凝素(HA)基因并组建了以HA为选择标记的系列表达载体,可用于不同结构和不同要求的外源基因在真核细胞中的表达。本系列载体的优点是,在病毒重组过程中可避开传代细胞系,一次性选择出可供疫苗使用的重组病毒。而HA阴性的重组病毒的毒力在家兔皮内试验中,未见明显下降。     

Isolation of a monoclonal antibody which blocks vaccinia virus infection.

We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry.