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Short Communication |
1 Institute of Virology, University of Leipzig, Leipzig, Germany
2 Department of Biology, Georgia State University, Atlanta, GA 30303, USA
Correspondence
Teryl K. Frey
tfrey{at}gsu.edu
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ABSTRACT |
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MAIN TEXT |
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). The 5'-terminal ORF (NS-ORF) encodes the non-structural proteins P150 and P90 and is translated from the genomic RNA as a polyprotein in the order NH2–P150–P90–COOH. Cleavage of P150 and P90 is mediated by a protease located at the C terminus of P150. These proteins undertake virus RNA synthesis in association with membranes of late endosomes and lysosomes (Magliano et al., 1998). The 3'-terminal ORF (SP-ORF) encodes the structural proteins and is translated from a subgenomic (SG) RNA as a polyprotein in the order NH2–capsid (C)–E2–E1–COOH. The RUB virion consists of the nucleocapsid and a lipid envelope in which the two envelope glycoproteins, E1 and E2, are embedded as a heterodimeric spike complex.
Superinfection exclusion or superinfection interference is a phenomenon common to most virus families by which an established virus infection blocks replication of a homologous superinfecting virus. Superinfection exclusion can be executed at different steps of the virus replication cycle, including attachment (Breiner et al., 2001a, b; Nethe et al., 2005; Schneider-Schaulies et al., 1995; Walters et al., 2004), penetration (Simon et al., 1990; Singh et al., 1997) and subsequent intracellular replication (Geib et al., 2003; Karpf et al., 1997; Lohmann et al., 2003; Schaller et al., 2007). Superinfection exclusion has also been studied with members of the alphaviruses, the other togavirus genus. Translation of the Sindbis virus non-structural proteins is sufficient to exclude superinfecting homologous virus (Adams & Brown, 1985) and the block occurs at an intracellular step, as replication of transfected Sindbis virus RNA is also restricted (Igarashi et al., 1977). During its maturation, nsP2 protease-mediated processing of the alphavirus non-structural precursor converts it first from an inactive enzyme to an active form that catalyses only minus-strand RNA synthesis, then to a form that catalyses both plus- and minus-strand synthesis and, finally, to the mature form that synthesizes only plus-strand RNA. It was thus suggested that the nsP2 protease of the resident virus is able to cleave the non-structural polyprotein precursor of the superinfecting virus prematurely, thereby terminating the replication of the superinfecting virus by inhibiting its ability to synthesize minus-strand RNA (Karpf et al., 1997).
RUB infection establishes resistance to superinfection with various heterologous viruses (Parkman et al., 1964), mediated through induction of interferon (Desmyter et al., 1968), which was the basis for the classical clinical assay for diagnosis of the virus (Carver et al., 1967). However, RUB-induced homologous superinfection exclusion has never been addressed. The asynchronous nature of RUB infection in cell culture (reviewed by Frey, 1994) complicates analysis of superinfection exclusion using RUB-infected cells. Cell cultures infected persistently by RUB are available (Abernathy et al., 1990), but it has been shown that the phenotype of superinfection exclusion can change in persistently infected cells (Lee et al., 2005). Therefore, we took advantage of the development of bicistronic RUB replicons expressing antibiotic-resistance genes (RUBrep/GFP/Neo; Fig. 1), through which cell cultures uniformly harbouring persistently replicating replicons can be selected and maintained (Fontana et al., 2007). Cells transfected with corresponding replicons of other viruses have recently been employed for analysis of superinfection exclusion (Lee et al., 2005; Sawicki et al., 2006; Tscherne et al., 2007). As shown in Fig. 2(a), plaque formation by RUB on cells infected persistently with this replicon (termed ‘GFP-RUB-Vero’ cells in this report) was reduced by tenfold and the plaques were smaller and opaque in comparison with plaques formed on Vero cells. Plaque formation by the heterologous Sindbis virus was similar on both Vero and GFP-RUB-Vero cells and, thus, homologous superinfection exclusion was at work. Superinfection exclusion among alphaviruses effects a decrease in titre from approximately 100-fold in vertebrate cells to 105-fold in mosquito cells (Adams & Brown, 1985; Karpf et al., 1997), in comparison with the approximately 1 log restriction apparent in RUB-induced superinfection exclusion. A similar diversity in the extent of restriction is observed among the flaviviruses: superinfection exclusion by bovine viral diarrhea virus is associated with a 105-fold decrease in titres of the superinfecting virus (Lee et al., 2005), whilst with hepatitis C virus, the decrease is only approximately tenfold (Tscherne et al., 2007).
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), which expresses red fluorescent protein (RFP) from the SG RNA and whose replication can thus be monitored by fluorescence microscopy of living cells. Packaged RUBrep/RFP particles were produced by transfecting a BHK cell line stably expressing the RUB SP-ORF (BHK-CE2E1) with RUBrep/RFP in vitro transcripts. When the same dilution of packaged RUBrep/RFP particles was used to infect Vero and GFP-RUB-Vero cells, the number of RFP-expressing cells in the GFP-RUB-Vero culture counted 2 days post-infection was only 20 % of that in Vero cells (data not shown), a restriction similar in magnitude to that observed in the number of plaques produced when these cells were infected with RUB (Fig. 2a). As shown in Fig. 2(b), superinfection exclusion was exhibited regardless of whether RUBrep/RFP was introduced by superinfection with packaged particles or ‘supertransfection’ with in vitro transcripts. As the structural proteins are not expressed in GFP-RUB-Vero cells, superinfection exclusion is mediated by the non-structural proteins.
Superinfection exclusion of RUBrep/RFP in GFP-RUB-Vero cells was exhibited following both superinfection and supertransfection and thus occurs at a step in the replication cycle after adsorption and entry. As shown in Fig. 3(a), Northern blotting was used to assess RNA synthesis by supertransfecting RUBrep/RFP transcripts; however, no RNAs specific to this replicon were detectable, indicating that replication was blocked at an early step in the replication cycle prior to significant RNA accumulation. Predictably, P150 and P90 synthesis was detectable in Vero cells 3 days post-transfection, but not in GFP-RUB-Vero cells (Fig. 3b). It has been shown that, similar to the alphaviruses, minus-strand RNA synthesis in RUB-infected cells is catalysed by the uncleaved non-structural precursor and cleavage is necessary for plus-strand RNA synthesis to proceed (Liang & Gillam, 2000, 2001). Given the lack of detectable RNA accumulation by the supertransfecting replicon in GFP-RUB-Vero cells, it was therefore next of interest to determine whether superinfection exclusion was mediated by cleavage of the non-structural precursor translated from the supertransfecting replicon by the resident NS protease. To this end, GFP-RUB-Vero cells were supertransfected with RUBrep-HA/RFP, a replicon expressing a haemagglutinin (HA)-tagged P150, and RUBrep-HA/RFP-GDD*, a replicon with a substitution of GDD to AAA in the RNA-dependent RNA polymerase, rendering it unable to replicate (Fig. 1). As shown in Fig. 3(c), processed, HA-tagged P150 was detectable in both Vero and GFP-RUB-Vero cells 6 h post-transfection with both replicons. In itself, this result shows that superinfection exclusion was not mediated at the level of translation of the transfecting RNA. However, when in vitro transcripts from RUBrep-HA/RFP C1152A, a mutant of one of the catalytic sites of the NS protease, were employed, cleavage was not observed in either Vero or GFP-RUB-Vero cells and, thus, trans-cleavage of the non-structural precursor translated from the supertransfecting replicon by the resident NS protease could not be detected.
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) and therefore active replication is required for superinfection exclusion to be elicited. We considered the alphavirus model in which superinfection exclusion is mediated by the resident nsP2 protease, which cleaves incoming non-structural precursors before minus-strand synthesis occurs (Adams & Brown, 1985; Igarashi et al., 1977; Karpf et al., 1997; Sawicki et al., 2006). However, we were unable to detect cleavage of an incoming non-structural precursor with an inactive NS protease. The results of a recent study using Semliki Forest virus and Sindbis virus suggested that inappropriate cleavage of non-structural precursors was not at work in modification of minus-strand RNA synthesis in cells infected with viruses and replicons expressing different minus-strand synthetic phenotypes (Sawicki et al., 2006); however, this study did not rule out the possibility that alphavirus-mediated superinfection exclusion is due to a restriction of minus-strand synthesis by the superinfecting virus. Therefore, RUB-induced superinfection exclusion could also be mediated at the minus-strand RNA synthesis step. Other models for superinfection exclusion at intracellular steps in the replication cycle invoke competition for cell functions or replication sites between the resident and superinfecting viruses (summarized by Karpf et al., 1997) and these are also consistent with our observations thus far on the RUB-induced version of the event.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Adams, R. H. & Brown, D. T. (1985). BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus. J Virol 54, 351–357.
Breiner, K. M., Schaller, H. & Knolle, P. A. (2001a). Endothelial cell-mediated uptake of a hepatitis B virus: a new concept of liver targeting of hepatotropic microorganisms. Hepatology 34, 803–808.[CrossRef][Medline]
Breiner, K. M., Urban, S., Glass, B. & Schaller, H. (2001b). Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes. J Virol 75, 143–150.
Carver, D. H., Marcus, P. I. & Seto, D. S. (1967). Intrinsic interference: a unique interference system used in assaying non-cytopathic viruses. Arch Gesamte Virusforsch 22, 55–60.[CrossRef][Medline]
Chen, M. H. & Icenogle, J. P. (2004). Rubella virus capsid protein modulates viral genome replication and virus infectivity. J Virol 78, 4314–4322.
Desmyter, J., Melnick, J. L. & Rawls, W. E. (1968). Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). J Virol 2, 955–961.
Fontana, J., Tzeng, W. P., Calderita, G., Fraile-Ramos, A., Frey, T. K. & Risco, C. (2007). Novel replication complex architecture in rubella replicon-transfected cells. Cell Microbiol 9, 875–890.[CrossRef][Medline]
Forng, R. Y. & Frey, T. K. (1995). Identification of the rubella virus nonstructural proteins. Virology 206, 843–853.[CrossRef][Medline]
Frey, T. K. (1994). Molecular biology of rubella virus. Adv Virus Res 44, 69–160.[Medline]
Geib, T., Sauder, C., Venturelli, S., Hassler, C., Staeheli, P. & Schwemmle, M. (2003). Selective virus resistance conferred by expression of Borna disease virus nucleocapsid components. J Virol 77, 4283–4290.
Igarashi, A., Koo, R. & Stollar, V. (1977). Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus. Virology 82, 69–83.[CrossRef][Medline]
Karpf, A. R., Lenches, E., Strauss, E. G., Strauss, J. H. & Brown, D. T. (1997). Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus. J Virol 71, 7119–7123.[Abstract]
Lee, Y. M., Tscherne, D. M., Yun, S. I., Frolov, I. & Rice, C. M. (2005). Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication. J Virol 79, 3231–3242.
Liang, Y. & Gillam, S. (2000). Mutational analysis of the rubella virus nonstructural polyprotein and its cleavage products in virus replication and RNA synthesis. J Virol 74, 5133–5141.
Liang, Y. & Gillam, S. (2001). Rubella virus RNA replication is cis-preferential and synthesis of negative- and positive-strand RNAs is regulated by the processing of nonstructural protein. Virology 282, 307–319.[CrossRef][Medline]
Lohmann, V., Hoffmann, S., Herian, U., Penin, F. & Bartenschlager, R. (2003). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77, 3007–3019.
Magliano, D., Marshall, J. A., Bowden, D. S., Vardaxis, N., Meanger, J. & Lee, J. Y. (1998). Rubella virus replication complexes are virus-modified lysosomes. Virology 240, 57–63.[CrossRef][Medline]
Marr, L. D., Wang, C. Y. & Frey, T. K. (1994). Expression of the rubella virus nonstructural protein ORF and demonstration of proteolytic processing. Virology 198, 586–592.[CrossRef][Medline]
Nethe, M., Berkhout, B. & van der Kuyl, A. C. (2005). Retroviral superinfection resistance. Retrovirology 2, 52[CrossRef][Medline]
Parkman, P. D., Buescher, E. L., Artenstein, M. S., McCown, J. M., Mundon, F. K. & Druzd, A. D. (1964). Studies of rubella. I. Properties of the virus. J Immunol 93, 595–607.
Pugachev, K. V. & Frey, T. K. (1998). Effects of defined mutations in the 5' nontranslated region of rubella virus genomic RNA on virus viability and macromolecule synthesis. J Virol 72, 641–650.
Sawicki, D. L., Perri, S., Polo, J. M. & Sawicki, S. G. (2006). Role for nsP2 proteins in the cessation of alphavirus minus-strand synthesis by host cells. J Virol 80, 360–371.
Schaller, T., Appel, N., Koutsoudakis, G., Kallis, S., Lohmann, V., Pietschmann, T. & Bartenschlager, R. (2007). Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol 81, 4591–4603.
Schneider-Schaulies, J., Schnorr, J. J., Brinckmann, U., Dunster, L. M., Baczko, K., Liebert, U. G., Schneider-Schaulies, S. & ter Meulen, V. (1995). Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains. Proc Natl Acad Sci U S A 92, 3943–3947.
Simon, K. O., Cardamone, J. J., Jr, Whitaker-Dowling, P. A., Youngner, J. S. & Widnell, C. C. (1990). Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus. Virology 177, 375–379.[CrossRef][Medline]
Singh, I. R., Suomalainen, M., Varadarajan, S., Garoff, H. & Helenius, A. (1997). Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus. Virology 231, 59–71.[CrossRef][Medline]
Tscherne, D. M., Evans, M. J., von Hahn, T., Jones, C. T., Stamataki, Z., McKeating, J. A., Lindenbach, B. D. & Rice, C. M. (2007). Superinfection exclusion in cells infected with hepatitis C virus. J Virol 81, 3693–3703.
Tzeng, W. P., Matthews, J. D. & Frey, T. K. (2006). Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue. J Virol 80, 3966–3974.
Walters, K. A., Joyce, M. A., Addison, W. R., Fischer, K. P. & Tyrrell, D. L. (2004). Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen. J Virol 78, 7925–7937.
Wang, X. & Gillam, S. (2001). Mutations in the GDD motif of rubella virus putative RNA-dependent RNA polymerase affect virus replication. Virology 285, 322–331.[CrossRef][Medline]
Received 16 April 2007;
accepted 2 July 2007.
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