Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 02 Aug 2019 17:19:35 Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2a Simone Backes, 1 3 Karin M. Sperling, 2 3 Joachim Zwilling, 2 3 Georg Gasteiger, 1 Holger Ludwig, 2 Elisabeth Kremmer, 3 Astrid Schwantes, 2 Caroline Staib 1 34 and Gerd Sutter 2,4 3 Correspondence Caroline Staib cstaib@genelux.com Gerd Sutter gerd.sutter@lmu.de 1 Institute of Virology, Technical University of Munich, and Helmholtz Center Munich, Germany 2 Division of Virology, Paul-Ehrlich-Institut, Langen, Germany 3 Institute for Molecular Immunology, Helmholtz Center Munich, Germany 4 Institute for Infectious Diseases and Zoonoses, University of Munich, Veterina ¨ rstrasse 13, 80539 Munich, Germany Received 24 July 2009 Accepted 15 October 2009 Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2a (eIF2a), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2a phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-DC7L) lacked late gene expression, which could be rescued by the function of host-range factor K1 or C7. It was demonstrated that viral gene expression was blocked after viral DNA replication and that it was independent of apoptosis induction. Furthermore, it was found that eIF2a phosphorylation in MVA-DC7L-infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts lacking PKR function, and it was shown that this was not due to reduced E3L gene expression. The block of eIF2a phosphorylation by C7 could be complemented by K1 in cells infected with MVA-DC7L encoding a reinserted K1L gene (MVA-DC7L-K1L). Importantly, these data illustrated that eIF2a phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and probably also for VACV replication in a diverse set of cell types. INTRODUCTION Modified vaccinia virus Ankara (MVA), a promising candidate replacement vaccine against smallpox, is cur- rently being investigated as a vector vaccine against infectious diseases and cancer (reviewed by Acres & Bonnefoy, 2008; Drexler et al., 2004; Go ´ mez et al., 2008). Its safety has been tested in over 120 000 humans (reviewed by Sutter & Staib, 2003). During the attenuation process, MVA has lost a substantial part of the vaccinia virus (VACV) genome sequence and is unable to replicate productively in most mammalian cells (Antoine et al., 1998; Carroll & Moss, 1997; Meyer et al., 1991). Nevertheless, MVA infection of non-permissive cells induces complete cascade-like gene expression, but is blocked in virion morphogenesis at the late stage of the viral life cycle (Sutter & Moss, 1992). Importantly for use as a vaccine, the inability to form infectious progeny does not affect induction of an efficient immune response against MVA- encoded recombinant and viral antigens (Sutter et al., 1994). VACV host tropism relies on the expression of certain host-range genes that enable virus replication (Werden et al., 2008). This gene class and the corresponding gene 3These authors contributed equally to this work. 4Present address: Genelux Corporation, Am Neuland 1, 82347 Bernried, Germany. Journal of General Virology (2010), 91, 470–482 DOI 10.1099/vir.0.015347-0 470 015347 G 2010 SGM Printed in Great Britain