Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Mice immunized with measles virus develop antibodies to a cell surface receptor for binding virus.

Mice were immunized with measles virus to determine whether an auto-anti-idiotypic antireceptor response could be generated as a probe for measles virus receptors. Mice initially responded to viral antigens (days 11 to 18) and subsequently developed antibodies to a putative measles virus receptor (peak at day 30 to 35) by three criteria: the sera (1) agglutinated erythrocytes which virus agglutinates, (2) reacted with Vero cells, and (3) inhibited virus attachment to Vero cells. Additionally, select sera inhibited virus infection of Vero cells. The cell-reactive activity was identified as immunoglobulin G antibody and was neutralized by sera reacting with virus (idiotype). The application of this anti-idiotypic antibody to identify measles virus-binding sites on Vero cells was revealed by the ability of sera to immunoprecipitate 20- and 30.5-kilodalton proteins from metabolically labeled ([35S]methionine) Vero cells.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Influence of the infection with lipid-containing viruses on the metabolism and pools of phospholipid precursors in animal cells

The influence of infection with three different lipid-containing RNA viruses, Newcastle disease virus, fowl plague virus, and Semliki Forest virus on the phosphatidylcholine precursors of chick embryo cells and of baby hamster kidney (BHK) cells has been measured. In chick embryo cells infection with Newcastle disease virus does not influence the energy charge, or the distribution and absolute pool sizes of the precursors or the choline phosphotransferase activity. In chick embryo cells infected with fowl plague virus the CDP-choline pool increases because of an inhibition of the choline phosphotransferase activity. The phosphorylcholine and CTP pools are smaller in infected cells when compared with mock-infected ones, although the energy charge is not influenced by infection. In chick embryo cells as well as in BHK cells the energy charge is diminished by infection with Semliki Forest virus. Therefore the CTP and phosphorylcholine pools are decreased. The CDP-choline pool in chick embryo cells becomes extremely small after infection with Semliki Forest virus because of a significant stimulation of the choline phosphotransferase. In BHK cells infected with Semliki Forest virus the opposite effect is observed. There are also severe effects on the uptake of the labeled precursors by infection. One and the same virus (Semliki Forest virus) has two completely different effects on the phosphatidylcholine precursors when infecting two different cell types. If one and the same cell type (chick embryo cells) is infected with three different lipid-containing RNA viruses also completely different effects on the phosphatidylcholine precursors were observed. Thus, each virus develops its own strategy to influence the lipid metabolism of the host cell, depending also on the choice of the host. This explains the many disturbing contradictory results described in the literature about the influence of lipid-containing viruses on the lipid metabolism of the host.     

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.     

Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.     

Glycoproteins of herpes simplex virus type 2 as defined by monoclonal antibodies.

We used monoclonal antibodies reacting with glycoproteins specified by herpes simplex virus type 2 (HSV-2) to characterize the individual antigens in terms of structure, processing, and kinetics of synthesis in BHK or Vero infected cells. Our results provided a direct demonstration of the structural identity of the gA and gB proteins of HSV-2 as well as confirmation of the existence of type-specific and type-common domains within the gD molecule. They also show that, with the exception of gC, processing of the viral glycoproteins differs to some extent in Vero and BHK infected cells, possibly as a result of different efficiency of glycosylation or different processing of underglycosylated and unglycosylated products in the two cell types. Finally, we showed that individual HSV-2 glycoproteins are synthesized at greatly different times during the infectious cycle, possibly in response to their different roles in virus replication and assembly.     

Inhibition of Sindbis virus maturation after treatment of infected cells with trypsin.

Brief treatment of Sindbis virus-infected BHK-21 or Vero cells with low concentrations of trypsin irreversibly blocked further production of progeny virions after removal of the enzyme. The inhibitory effects of the trypsin treatment could only be demonstrated in cells in which virus infection was established; optimal inhibition occurred at ca. 3 h postinfection. Production of virus structural proteins PE2, E1, and C occurred at normal levels in inhibited cells. PE2 and E1 were also transported to the cell plasma membrane during inhibition; however, PE2 was not cleaved to E2, and little capsid protein became membrane associated relative to control cells. Although trypsin treatment had no effect on Sindbis protein synthesis, the production of both 26S and 42S RNA was greatly reduced. Similar trypsin treatment of BHK cells infected with vesicular stomatitis virus had no detectable effect on the course of virus infection.     

Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.

Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH.     

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.     

The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis

It has previously been shown that upon infection of HeLa cells with modified vaccinia virus Ankara (MVA), assembly is blocked at a late stage of infection and immature virions (IVs) accumulate (G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). In the present study the morphogenesis of MVA in HeLa cells was studied in more detail and compared to that under two conditions that permit the production of infectious particles: infection of HeLa cells with the WR strain of vaccinia virus (VV) and infection of BHK cells with MVA. Using several quantitative and qualitative assays, we show that early in infection, MVA in HeLa cells behaves in a manner identical to that under the permissive conditions. By immunofluorescence microscopy (IF) at late times of infection, the labelings for an abundant membrane protein of the intracellular mature virus, p16/A14L, and the viral DNA colocalize under permissive conditions, whereas in HeLa cells infected with MVA these two structures do not colocalize to the same extent. In both permissive and nonpermissive infection, p16-labeled IVs first appear at 5 h postinfection. In HeLa cells infected with MVA, IVs accumulated predominantly outside the DNA regions, whereas under permissive conditions they were associated with the viral DNA. At 4 h 30 min, the earliest time at which p16 is detected, the p16 labeling was found predominantly in a small number of distinct puncta by IF, which were distinct from the sites of DNA in both permissive and nonpermissive infection. By electron microscopy, no crescents or IVs were found at this time, and the p16-labeled structures were found to consist of membrane-rich vesicles that were in continuity with the cellular endoplasmic reticulum. Over the next 30 min of infection, a large number of p16-labeled crescents and IVs appeared abruptly under both permissive and nonpermissive conditions. Under permissive conditions, these IVs were in close association with the sites of DNA, and a significant amount of these IVs engulfed the viral DNA. In contrast, under nonpermissive conditions, the IVs and DNA were mostly in separate locations and relatively few IVs acquired DNA. Our data show that in HeLa cells MVA forms normal DNA replication sites and normal viral precursor membranes but the transport between these two structures is inhibited.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.     

Induction of p53-dependent and mitochondria-mediated cell death pathway by dengue virus infection of human and animal cells

Previous studies have suggested that cells undergo apoptosis in response to dengue virus infection. However, the potential significance of dengue virus-induced apoptosis and the pathways are still not clearly defined. In this study, comparative analysis of dengue virus-induced apoptosis in BHK, H1299, HUH-7 and Vero cell lines was carried out. We show here that infection of BHK, HUH-7 and Vero cell lines with dengue type 1 virus (DEN1V) induces cell death typical of apoptosis. Virus-induced cell death was assayed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, detection of oligonucleosomal DNA fragmentation, DNA content analysis and assay for the externalization of phosphatidylserine residues. Detailed study of dengue virus infection in HUH-7 cells showed activation of cell death via the mitochondrial pathway causing lowering of mitochondrial transmembrane potential (DeltaPsim) in HUH-7 cells. Interestingly, in the p53-deficient cell line, H1299, apoptosis was largely undetectable compared with the other cell lines used; suggesting that a p53- and mitochondria-mediated cell death pathway may play an important role in dengue virus-induced apoptosis.     

4-Deoxy-4-fluoro-D-mannose inhibits the glycosylation of the G protein of vesicular stomatitis virus

The effect of 4-deoxy-4-fluoro-D-mannose (4F-Man), a synthetic analog of D-mannose, on the synthesis of the glycoprotein (G) of vesicular stomatitis virus was examined. Nearly confluent monolayers of cultured BHK21 cells infected with vesicular stomatitis virus were incubated for 2 h with 4F-Man (0-10 mM) or for 1 h with tunicamycin (2 micrograms/ml) and then pulse-labeled with [35S]methionine or [3H]glucosamine. After a 90-min chase period, the cells were lysed and the viral proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The 35S-labeled G protein from cells exposed to greater than or equal to 1 mM 4F-Man migrated more rapidly than G protein isolated from control cells and with the same electrophoretic mobility as the glycoprotein produced by cells treated with tunicamycin. When infected cells were labeled with [3H]glucosamine, little or no radioactivity was associated with G protein synthesized in the presence of greater than or equal to 1 mM 4F-Man. The conclusion that 4F-Man blocks the glycosylation of the G protein was supported by experiments which demonstrated that the fluorosugar inhibits the synthesis of lipid-linked oligosaccharides.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Primate gammaretroviruses require an ancillary factor not required for murine gammaretroviruses to infect BHK cells

BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells

More than 50 RNAs expressed by Epstein-Barr virus late in productive infection have been identified. B95-8-infected cells were induced to a relatively high level of permissive infection with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate. Polyadenylated RNAs were extracted from the cell cytoplasm, separated by size on formaldehyde gels, transferred to nitrocellulose, and hybridized to labeled recombinant Epstein-Barr virus DNA fragments. Comparison of RNAs from induced cultures with RNAs from induced cultures also treated with phosphonoacetic acid to inhibit viral DNA synthesis identifies two RNA classes: a persistent early class of RNAs whose abundance is relatively resistant to viral DNA synthesis inhibition and a late class of RNAs whose abundance is relatively sensitive to viral DNA synthesis inhibition. The persistent early and late RNAs are not clustered but are intermixed and scattered through most of segments UL and US. The cytoplasmic polyadenylated RNAs expressed during latent infection were not detected in productively infected cells, indicating that different classes of viral RNA are associated with latent and productive infection. Non-polyadenylated small RNAs originally identified in cells latently infected with Epstein-Barr virus are expressed in greater abundance in productively infected cells and are part of the early RNA class.