Cellular laminin-binding protein and Venezuelan equine encephalomyelitis virus replication in Vero, 293 and 9S2 cells

The expression of the laminin-binding protein (LBP) on cellular membranes in different cell lines has been studied. A high level of replication of Venezuelan equine encephalomyelitis (VEE) virus was registered in Vero cells with high levels of LBP on the cell surface. The treatment of Vero cells with monoclonal antibodies to human LBP reduced VEE virus replication by a factor of more than 200. A low level of LBP expression on the surface of 293 cells was increased via transfection by plasmid with gene for human LBP. The VEE virus replication in transfected cells (9S2) was increased by more that 2000 times compared to the 293 cells. The results demonstrated the principal role of cellular LBP in the entry of VEE virus into mammalian cells. It is proposed that LBP is a key cellular protein for the early stage of the VEE virus replication in cells. LBP may be a target protein for the development of a new generation of antiviral drugs capable of inhibiting (enhancing) the alphavirus replication in human cells.     

Laminin-binding protein as a cellular receptor for the equine Venezuelan encephalomyelitis virus: Report 2. Inhibition of replication of equine Venezuelan encephalomyelitis virus by blocking laminin-binding protein on the surface of Vero cells

A possible inhibition of the Venezuelan equine encephalomyelitis (VEE) virus replication in Vero cells through the laminin-binding protein (LBP) blocking in the surface of such cells was investigated in order to verify the LBP value. It was demonstrated, on the basis of the flow scanning cytometry and FITC-labeled antibodies to LBP, that there are at least 263 thousand LBP molecules in the Vero cells' surface. Blocking of the molecules by rabbit polyclonal antibodies to 43 kD of the recombinant LBP (rLBP) was shown to inhibit the VEE virus replication in Vero cells by more than 300,000 times, which made them virtually resistant to the possibility of VEE virus infection. This also confirmed that LBP is a target-molecule for VEE virus in Vero cells with the interplay between VEE virus and LBP in the cells' surface being the initial stage of virions' penetration into the cell and of their replication inside the cell.     

A putative receptor for Venezuelan equine encephalitis virus from mosquito cells.

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.     

Recombinant wild-type and edmonston strain measles viruses bearing heterologous H proteins: role of H protein in cell fusion and host cell specificity

Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.     

A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICP0, a transactivating protein of herpes simplex virus type 1.

ICP0 transactivates herpes simplex virus type 1 genes of all classes as well as a number of heterologous viral and cellular genes, yet it is not essential for virus replication in vitro or in vivo. Stocks of ICP0 deletion mutants, however, exhibit significantly lower plating efficiencies on standard 24-h-old Vero cell monolayers than do stocks of wild-type virus. In an attempt to determine whether the growth status of cells in the monolayer affects the ability of ICP0 mutants to initiate plaque formation, the plating efficiencies and abilities of an ICP0 null mutant (7134) and of wild-type virus (KOS) to express selected viral proteins were determined on Vero cell monolayers whose growth had been arrested either by contact inhibition-trypsinization or by isoleucine deprivation and had then been released from growth arrest. The proportion of cells cycling synchronously after release from growth arrest was assessed by flow cytometry. The results of these studies indicate that the plating efficiency of 7134 was greatest on Vero cell monolayers 8 h after release from growth arrest induced by either treatment. Monolayers of both types released from growth arrest at other times supported 7134 plaque formation less efficiently. In contrast, the plating efficiency of KOS was nearly equal on monolayers at all times after release from growth arrest. Notably, both KOS and 7134 were equally efficient in entering cells and inducing expression of the immediate-early protein ICP4 in either 8- or 24-h monolayers. Relative to KOS, however, 7134 was significantly impaired in the expression of selected early and late genes in cells at 24 h postrelease. When the plating efficiencies of 7134 and KOS were examined in 0-28 cells (Vero cells that are stably transformed with the ICP0 gene) whose growth had been arrested and then released, no differences in the plating efficiencies of the two viruses as a function of growth status were noted. These findings suggest that a cellular function expressed maximally in cells 8 h after release from growth arrest can substitute operationally for ICP0 to enhance plaque formation and viral gene expression by 7134. They further suggest that one role of ICP0 in viral infection is to facilitate virus replication in cells that do not express this function.     

Laminin-binding protein (LBP) as a cellular receptor for the virus of Venezuelan equine encephalomyelitis (VEE): Part 1. A study of the interaction between VEE virus virions and the human recombinant LBP

The interaction of the VEE virus virions with human LBP was investigated. The affinely purified 43 kDa recombinant LBP (rLBP) of man was found to interact effectively with the VEE virus virions purified in immune enzyme assay. The affinity constant of 43 kDa rLBP with virions was equal to 4.3-4.8 x 10(7) M-1. The rabbit antiviral polyclonal antibodies blocked the interaction of rLBP with the VEE virus virions. According to Western blot, rLBP is capable of interacting with the E1 glycoprotein of the VEE virus, which suggests the presence of a specific epitope of binding with LBP in the surface of the E1-E2 heterodimer of the VEE virus. The results confirm that human LBP could be a receptor for the VEE virus.     

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.     

表达登革病毒prM基因小干扰RNA的Vero细胞系的建立

prM蛋白是登革病毒膜蛋白M的前体,膜蛋白M对病毒的组装与成熟有重要作用,针对prM基因设计的小干扰RNA（siRNA）可短期抑制登革病毒复制.为了达到长期抑制登革病毒的效果,本研究构建了插入prM siRNA序列的重组慢病毒,利用流式细胞术分选以及杀稻瘟霉素抗性,筛选出稳定表达prM siRNA的非洲绿猴肾细胞（Vero细胞）系.经逆转录PCR及测序验证siRNA序列表达正确. Vero细胞中prM siRNA的表达率约为976%.当受到登革病毒攻击时,表达prM siRNA的Vero细胞能够明显抑制登革病毒prM基因的表达,并抑制登革病毒在Vero细胞中的复制.建立的Vero细胞系可用于RNA干扰防治登革病毒感染的进一步应用研究.     

Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses

The Edmonston strain of measles virus (MV) was obtained by sequential passages of the original isolate in various cultured cells. Although attenuated in vivo, it grows efficiently in most primate cell lines. Previous studies have revealed that MV tropism cannot be solely explained by the use of CD150 and/or CD46 as a cellular receptor. In order to evaluate the contributions of individual genes of the Edmonston strain to growth in cultured cells, we generated a series of recombinant viruses in which part of the genome of the clinical isolate IC-B (which uses CD150 as a receptor) was replaced with the corresponding sequences of the Edmonston strain. The recombinant virus possessing the Edmonston hemagglutinin (H) gene (encoding the receptor-binding protein) grew as efficiently in Vero cells as the Edmonston strain. Those viruses having either the matrix (M) or large (L) protein gene from the Edmonston strain could also replicate well in Vero cells, although they entered them at low efficiencies. P64S and E89K substitutions were responsible for the ability of the M protein to make virus grow efficiently in Vero cells, while the first half of the Edmonston L gene was important for better replication. Despite efficient growth in Vero cells, the recombinant viruses with these mutations had growth disadvantage in CD150-positive lymphoid B95a cells. Thus, not only the H gene but also the M and L genes contribute to efficient replication of the Edmonston strain in some cultured cells.     

Molecular integrity and usefulness of episomal expression vectors derived from BK and Epstein-Barr virus

High-level and stable production of a protein of interest is one of the most important parameters when considering the development of an efficient vector system for heterologous gene expression. In order to achieve this goal, we have used episomal vector elements derived from Epstein-Barr virus (EBV) or BK virus (BKV) in combination with the strictly regulated interferon-inducible Mx promoter.Here we demonstrate that EBV-derived vectors replicate efficiently in all cell lines tested (i.e. HEK293, HeLaH21 and Vero), yielding stable transfectants with a high, inducible expression level and almost no background. In contrast, BKV-derived vectors are much more restricted to particular cell types and hampered by DNA rearrangements, which is a serious drawback for use over a longer timespan.     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

Interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication

The human cytomegalovirus UL82-encoded pp71 protein is required for efficient virus replication and immediate-early gene expression when cells are infected at a low multiplicity. Functions attributed to pp71 include the ability to enhance the infectivity of viral DNA, bind to and target hypophosphorylated Rb family member proteins for degradation, drive quiescent cells into the cell cycle, and bind to the cellular protein hDaxx. Using UL82 mutant viruses, we demonstrate that the LXCXD motif within pp71 is not necessary for efficient virus replication in fibroblasts, suggesting that pp71's ability to degrade hypophosphorylated Rb family members and induce quiescent cells into the cell cycle is not responsible for the growth defect associated with a UL82 deletion mutant. However, UL82 mutants that cannot bind to hDaxx are unable to induce immediate-early gene expression and are severely attenuated for viral replication. These results indicate that the interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.     

The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence

Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.     

Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.     

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.     

Vaccinia virus-based expression of gp120 and EGFP: survey of mammalian host cell lines

Production of recombinant proteins with the vaccinia virus expression system in five mammalian cell lines (HeLa, BS-C-1, Vero, MRC-5, and 293) was investigated for protein yield and proper posttranslational modifications. Regulatory acceptance of the host cell line was taken into consideration, where Vero, MRC-5, and 293 were considered more acceptable to the regulatory authorities. Relevant process knowledge for ease of scale-up with the particular cell type was also considered. Two proteins were expressed, enhanced green fluorescent protein (EGFP) in the cytoplasm and gp120, an HIV envelope coat protein that is secreted into the culture medium. HeLa cells produced the most EGFP at 17.2 microg/well with BS-C-1 and 293 following. BS-C-1 produced the most gp120 at 28.2 microg/mL with 293 and Vero following. Therefore, of the three most appropriate cell lines (Vero, MRC-5, and 293) for production processes, the best results were obtained with 293 cells. Although MRC-5 had a very high productivity on a per cell basis, the low cell density and slow growth rate made the overall production insufficient. Because gp120 contained a significant amount of posttranslational modification, this protein, produced by the different cell lines, was further analyzed by PNGase digestion suggesting N-linked glycosylation modifications in all cell lines tested. On the basis of these results and overall process considerations, 293 cells are recommended for further production process optimization in a serum-free suspension system.