Observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox

Ectromelia virus was shown to replicate in vitro in all lymphoma cell lines and in a small proportion of hybridoma lines tested. It was demonstrated that certain hybridoma cell lines, which were passed in ectromelia virus-infected mice, yielded ectromelia virus infectivity on explantation into tissue culture. This finding further substantiated the belief that ascitic fluid and hybridoma cell lines exposed to virus during mouse-passage could be important in the epidemiology of mousepox.     

Genetic analysis of the herpes simplex virus type 1 UL20 protein domains involved in cytoplasmic virion envelopment and virus-induced cell fusion

The herpes simplex virus type 1 UL20 protein (UL20p) is an important determinant for cytoplasmic virion morphogenesis and virus-induced cell fusion. To delineate the functional domains of the UL20 protein, we generated a panel of single and multiple (cluster) alanine substitutions as well as UL20p carboxyl-terminal truncations. The UL20 mutant genes could be broadly categorized into four main groups: Group I UL20 mutant genes complemented for both virus production and virus-induced cell fusion; Group II UL20 mutant genes did not complement for either virus-induced cell fusion or infectious virus production; Group III UL20 mutant genes complemented for virus-induced cell fusion to variable extents but exhibited substantially decreased ability to complement UL20-null infectious virus production; Group IV mutant genes complemented for infectious virus production but had variable effects on virus-induced cell fusion; this group included two mutants that efficiently complemented for gBsyn3, but not for gKsyn1, virus-induced cell fusion. In addition, certain recombinant viruses with mutations in either the amino or carboxyl termini of UL20p produced partially syncytial plaques on Vero cells in the absence of any other virally encoded syncytial mutations. These studies indicated that the amino and carboxyl termini of UL20p contained domains that functioned both in infectious virus production and virus-induced cell fusion. Moreover, the data suggested that the UL20p's role in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis and that certain UL20p domains that function in gB-syn3 virus-induced cell fusion are distinct from those functioning in gKsyn1 virus-induced cell fusion.     

病毒治癌

病毒治疗癌症近来有新进展．分子生物学技术的发展,大大促进了病毒在寄主专一性、基因传递的定位性及溶解癌细胞方面的研究：选择适当的病毒种类,根据癌细胞特征性表面蛋白的特性修饰病毒的表面蛋白,使之对癌细胞具有更强的亲和性;去除病毒中原有的致病基因如癌基因,并根据靶细胞的特性针对性地改造有关病毒基因如插入适当的抗癌基因,可能构建成安全有效的工程病毒用于癌症治疗．病毒治癌除具有较强的特异性外,和其他基因治疗方法相比,还具有能利用寄主细胞进行自我复制、级联放大以克服初始剂量不足等特点．     

Herpes simplex virus: receptors and ligands for cell entry

Entry of herpes simplex virus (HSV) into cells depends upon multiple cell surface receptors and multiple proteins on the surface of the virion. The cell surface receptors include heparan sulphate chains on cell surface proteoglycans, a member of the tumor necrosis factor (TNF) receptor family and two members of the immunoglobulin superfamily related to the poliovirus receptor. The HSV ligands for these receptors are the envelope glycoproteins gB and gC for heparan sulphate and gD for the protein receptors and specific sites in heparan sulphate generated by certain 3-O-sulfotransferases. HSV gC also binds to the C3b component of complement and can block complement-mediated neutralization of virus. The purposes of this review are to summarize available information about these cell surface receptors and the viral ligands, gC and gD, and to discuss roles of these viral glycoproteins in immune evasion and cellular responses as well as in viral entry.     

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.     

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.     

Model-driven approaches for in vitro combination therapy using ONYX-015 replicating oncolytic adenovirus

Replicating genetically modified adenoviruses have shown promise as a new treatment approach against cancer. Recombinant adenoviruses replicate only in cancer cells which contain certain mutations, such as the loss of functional p53, as is the case in the virus ONYX-015. The successful entry of the viral particle into target cells is strongly dependent on the presence of the main receptor for adenovirus, the coxsackie- and adenovirus receptor (CAR). This receptor is frequently down-regulated in highly malignant cells, rendering this population less vulnerable to viral attack. It has been shown that the use of MEK inhibitors can up-regulate CAR expression, resulting in enhanced adenovirus entry into the cells. However, inhibition of MEK results in G1 cell cycle arrest, rendering infected cells temporarily unable to produce virus. This forces a tradeoff. While drug mediated up-regulation of CAR enhances virus entry into cancer cells, the consequent cell cycle arrest inhibits production of new virus particles and the replication of the virus. Optimal control-based schedules of MEK inhibitor application should increase the efficacy of this treatment, maximizing the overall tumor toxicity by exploiting the dynamics of CAR expression and viral production. We introduce a mathematical model of these dynamics and show simple optimal control based strategies which motivate this approach.     

Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 plaque production was inhibited by treating cells with soluble forms of HSV-1 glycoprotein D (gD-1t) and HSV-2 glycoprotein D (gD-2t). Both glycoproteins inhibited entry of HSV-1 and HSV-2 without affecting virus adsorption. In contrast, a soluble form of HSV-2 glycoprotein B had no effect on virus entry into cells. Specific binding of gD-1t and gD-2t to cells was saturable, and approximately 4 x 10(5) to 5 x 10(5) molecules bound per cell. Binding of gD-1t was markedly reduced by treating cells with certain proteases but was unaffected when cell surface heparan sulfate glycosaminoglycans were enzymatically removed or when the binding was carried out in the presence of heparin. Together, these results suggest that gD binds to a limited set of cell surface receptors which may be proteins and that these interactions are essential for subsequent virus entry into cells. However, binding of gD to its receptors is not required for the initial adsorption of virus to the cell surface, which involves more numerous sites (probably including heparan sulfate) than those which mediate gD binding.     

狂犬病病毒糖蛋白重组人腺病毒的构建及免疫效果的初步研究

构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。     

Defective bud formation in human cells chronically infected with subacute sclerosing panencephalitis virus.

Human prostate cells chronically infected with the Mantooth strain of subacute sclerosing panencephalitis (SSPE) virus multiply normally, fuse only occasionally to form giant cells, and yet have twisted intracytoplasmic nucleocapsids. These cells are able to support replication of vesicular stomatitis virus, although they release only small amounts of SSPE virus. To determine why carrier cells do not produce virus, they were examined with techniques for surface replication, freeze-fracturing, and immunoperoxidase labeling with SSPE antibody. The surface of carrier cells, like that of productive cells, is characterized by ridges crowned with viral antigens and devoid of the intramembrane particles revealed by freeze-fracture techniques. Since surface ridges form where nucleocapsids attach to the membrane, the shape and length of ridges are indicative of the shape and length of the underlying nucleocapsid. Whereas ridges on productive cells are serpentine in shape, those on carrier cells are typically straight or hairpin shaped, and the hairpin ridges are twice as long as serpentine ridges on productive cells. Furthermore, the spacing between ridges on carrier cells is never as small as that in productive infections, so that continuous sheets of viral membrane are never formed. The majority of carrier cells lack the round viral buds observed in productive cells but have, instead, many elongated processes attached to the cell surface. Each of these processes contains one or two hairpin ridges overlying hairpin-shaped nucleocapsids. These "hairpin buds" are restricted to a single region of the carrier cell surface, whereas viral buds are distributed over the entire surface of productive cells. Thus, there are several structural defects in carrier cells that depend on the specific interaction of a certain viral strain with a certain cell type. These defects prevent the deployment of viral antigen in some regions of the cell surface, the formation of nucleocapsids of normal length, the coiling of attached nucleocapsids, and the consolidation of sheets of viral membrane into spherical buds with the nucleocapsids coiled inside. These defects may account for the failure of carrier cells to shed infectious virus.     

Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles.

A highly sensitive single-round infection assay using a bacterial chloramphenicol acetyltransferase was developed to analyze an early stage of human immunodeficiency virus type 1 replication. By a combination of transfection and single-round infection assay, a virus with a vif mutation, depending on host cells from which the virus was derived, was demonstrated to be defective at the early phase of infection cycle. Analysis of viral proteins synthesized in cells indicated that incorporation of the Env surface protein into virions of the vif mutant, again in a cell-dependent way, was greatly restricted. Taken together, it is concluded that the Vif protein acts through modulation of the Env protein in the virions, directly or indirectly, to enhance viral infectivity in a certain cell type.     

Replacement of mouse LM fibroblast choline by a sulfonium analog. Effects on membrane properties as determined by virus probes

A sulfonium analog of choline ('sulfocholine', a natural phospholipid constituent of diatoms) was metabolically incorporated into mouse LM fibroblasts cultured in serum-free medium. Subconfluent cultures of LM cells were able to utilize sulfocholine as sole choline source and to increase in cell number for 3 days of incubation; thereafter a decrease in cell number was observed. In contrast, cultures of LM cells seeded to confluency showed no decrease in cell number up to at least 10 days when maintained, with daily medium changes, in medium containing either choline or the sulfonium analog. Such confluent cultures, maintained for 7 days in sulfocholine-containing medium, showed virtually complete replacement of cellular phosphatidylcholine and greater than 50% replacement of cellular sphingomyelin by their respective sulfonium analogs. The functional exchangeability of natural phosphatidylcholine and sphingomyelin with their sulfonium analogs to participate in normal cell membrane-mediated activities was demonstrated by comparatively assaying the abilities of sulfocholine- and choline-maintained cells to incorporate and replicate certain animal viruses known to possess membrane-dependent steps in various phases of their replication cycles. No difference was detected between the abilities of sulfocholine- and choline-maintained cells to take up vesicular stomatitis virus or mengo virus, or to replicate vesicular stomatitis virus, mengo virus or mouse hepatitis virus.     

Human lung carcinoma (A-549) continuing cell line and human endothelial (ECV-304) continuing cell line responses to the influenza virus at different multiplicities of infection

The course of infection upon virus entry into the cell depends not only on the biological characteristics of the cells and of the virus itself, but also on the intensity of the cell infection by the virus, i.e., on the multiplicity of infection. The purpose of our work was to perform a comparative study of the responses of two human cell lines, the lung carcinoma cell line A-549 and the endothelium cell line ECV-304, to the infection with the influenza virus A at different multiplicities of infection. At the first passage, both cell lines responded by enhancement of proliferation and apoptosis induction only to the low doses of influenza virus (ID 1–10). In A-49 cells, the stimulatory effect of the low virus doses was observed 1–2 days earlier than in ECV-304 cells. Enhanced proliferation was observed in both cell lines from the second to the fourth passages, when cells were infected with higher virus doses (ID 100 and 1000). In addition, the response of the A-549 cells to low doses of the H3N2 strain of the influenza virus A depended on the virus propagation conditions—namely, no enhancement of cell proliferation was observed in response to the infection with the virus propagating in chicken embryonated eggs, in contrast to infection with the virus that propagated in cell culture. Immunocytochemistry of A-549 cells has demonstrated that, on the third day after infection, there could be observed a change (in the dose-dependent manner) in the intracellular localization of p53 and cyclin A, proteins involved in the cell cycle progression. At the low virus dose, cyclin A was predominantly detected in the nuclei (63%), while at the high virus dose it was p53 (54%), which was predominantly detected in this cellular compartment, this observation confirming that stimulation of cell proliferation in the case of very low multiplicity of infection and cell division arrest takes place in the case of high multiplicity of influenza virus infection. The study of the influenza virus A reproduction in A-549 and ECV-304 cells using a whole number of virology techniques showed low sensitivity of these cells to the influenza virus, which manifested in the gradual decrease in the viral RNA expression and the impairment of mature viral particles assembly during several passages. Therefore, the decrease in the multiplicity of infection is associated in the A-549 and ECV-304 cells with impairment of production of mature virus particles or certain virus protein synthesis, which is accompanied by cell proliferation enhancement and apoptosis induction. As a result of the comparative study of the two cell lines (A-549 and ECV-304) upon infection with different doses of influenza virus A, we have revealed common principles and specific features indicating the effects of the biological properties of the viruses and cells, as well as of the multiplicity of infection on the course of virus infection.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Intracellular Synthesis of Myxovirus Neuraminidase in Chick Embryo Cell Monolayer Culture I. Neuraminidase Activity, Hemagglutinin Synthesis, and Content of Cell-bound Sialic Acid in Newcastle Disease Virus-infected Cells

Neuraminidase (Nase) activity of chick embryo monolayer cell homogenates was determined by its rate of splitting of neuraminlactose, free neuraminic acid (NA) being determined by the thiobarbituric acid assay. Noninfected cells were found to have no detectable amount of Nase activity. Newcastle disease virus (NDV)-infected cells (multiplicity of infection, 20 to 75 plaque-forming units per cell) displayed a high level of Nase synthesis, the rate of synthesis being parallel to that of hemagglutinin (HA) synthesis (with a 1.5 hr delay in the latter). An "eclipse" of the Nase and HA activities associated with the virus that was adsorbed onto cells was observed. The data provide evidence that the Nase is not incorporated into the viral envelope from a pre-existing cell supply but that its synthesis is coded by the viral genome. The content of cell-bound sialic acid, determined simultaneously in infected-cell homogenates, showed characteristic features allowing certain conclusions concerning the renewal of NA-terminating cell receptors during the course of infection, and the intracellular action of the Nase of the virus introduced into cells by the inoculum and that of the newly synthesized Nase at different stages of infection.     

Cell death signalling in virus infection

Apoptosis, necroptosis and pyroptosis represent three major regulated cell death modalities. Apoptosis features cell shrinkage, nuclear fragmentation and cytoplasm-blebbing. Necroptosis and pyroptosis exhibit osmotic imbalances in the cell accompanied by early membrane ruptures, which morphologically resembles necrosis. Importantly, these two lytic cell death forms facilitate the release of damage associated molecular patterns into the extracellular space leading to inflammatory response. Whereas, during apoptosis, the membrane integrity is preserved and the apoptotic cell is removed by neighbouring cells ensuring the avoidance of immune-stimulation. Viruses comprise a versatile group of intracellular pathogens, which elicit various strategies to infect and to propagate. Viruses are recognized by a myriad of pathogen recognition receptors in the human cells, which consequently lead to activation of the immune system and in certain circumstances cell-autonomous cell death. Importantly, the long-standing view that a cell death inducing capacity of a virus is equal to its pathogenic potential seems to be only partially valid. The altruistic cell death of an infected cell may serve the whole organism by ultimately curbing the way of virus manufacturing. In fact, several viruses express “anti-cell death” proteins to avoid this viral-defence mechanism. Conversely, some viruses hijack cell death pathways to selectively destroy cell populations in order to compromise the immune system of the host. This review discusses the pros and cons of virus induced cell death from the perspective of the host cells and attempts to provide a comprehensive overview of the complex network of cell death signalling in virus infection.     

Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent.

CXCR4 (also termed fusin, LESTR, or HUMSTR) is a member of the G-protein-coupled chemokine receptor family with seven membrane-spanning domains. CXCR4 acts as a coreceptor for syncytium-inducing human immunodeficiency virus type 1 (HIV-1) strains, conferring entry into CD4+ cells. We show here that a novel mouse monoclonal antibody (12G5) that recognizes CXCR4 blocked cell-to-cell fusion and cell free-virus infection of CXCR4+ CD4+ RD rhabdomyosarcoma cells by seven HIV-1 and HIV-2 strains that had various cell tropisms for different CD4+ human cell types. Yet the majority of the members of the same virus panel resisted 12G5 inhibition on T-cell lines. When inhibition was observed on these cell types, it was both cell type and virus strain dependent. In at least one situation, 12G5 failed to block LAI infection of cells expressing CXCR4 as the only available coreceptor. Our observations suggest that CXCR4 could be processed or presented differently depending on the cell type, allowing some strains to evade 12G5 inhibition. Alternatively, since several of the viruses could infect certain CXCR4- CD4+ cell lines, it is conceivable that alternative coreceptors are active, enabling individual HIV strains to choose between compatible coreceptors during entry into cells. Moreover, the strain dependency of 12G5 inhibition implies that the interaction of different HIVs with CXCR4 varies.     

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

Evidence of selective processing of immunodominant epitopes in virally infected cells

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.