Download Murine gammaherpesvirus 68 open reading frame 35 is required for

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Murine gammaherpesvirus 68 open reading frame 35 is required for efficient lytic
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replication and latency
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Running title: Characterization of MHV68 ORF35
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The Contents Category for the paper: Animal Viruses – Large DNA
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Shin-ichi Hikita, Yusuke Yanagi, and Shinji Ohno
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Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582,
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Japan
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Mailing address
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Shinji Ohno
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Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582,
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Japan; TEL: +81-92-642-6138 / FAX: +81-92-642-6140;
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E-mail: [email protected]
Corresponding author:
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Summary: 231 words, Text: 4872 words,
Tables and figures : 7
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SUMMARY
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Murine gammaherpesvirus (MHV) 68, a natural pathogen of field mice, is related to
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human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated
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herpesvirus (KSHV). The open reading frame (ORF) 35 of MHV68 and its homologues
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of EBV and KSHV are located in the gene cluster composed of ORF34 to ORF38 in
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which each gene overlaps with adjacent ones. Although MHV68 ORF35 was reported to
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be an essential gene, its function during infection is presently unknown. In this study, we
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show, by analyzing ORF35-transfected cells, that three serine residues in the C-terminus
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are responsible for the phosphorylation, and that the ORF35 protein forms
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homo-oligomers via a predicted coiled-coil motif. The ORF35 protein expressed by
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transfection was preferentially located in the cytoplasm of cells uninfected or infected
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with MVH68. The recombinant virus lacking ORF35 (35S virus) exhibited the genome
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replication and expression of lytic proteins comparable to those of the wild-type (WT)
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virus, but reduced levels of virus production, suggesting that the ORF35 protein acts at
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the virion assembly and/or egress step. Lytic replication in the lung after intranasal
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infection and the frequency of ex vivo reactivation from latency after intraperitoneal
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infection were lower in 35S virus-infected mice than those in mice infected with the WT
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or marker-reverted virus. Our results indicate that ORF35 is not essential for MHV68
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lytic replication, but plays an important role in efficient viral replication and reactivation
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from latency.
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INTRODUCTION
Human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma
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associated virus (KSHV), are involved in a number of malignant diseases. EBV causes
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Burkitt's lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, and gastric
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carcinoma (Burke et al., 1990; Epstein et al., 1964; Longnecker et al., 2013; Nonoyama
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et al., 1973; Weiss et al., 1987), whereas KSHV is a causative agent of Kaposi's sarcoma,
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multicentric Castleman's disease, and primary effusion lymphoma (Chang et al., 1994;
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Damania & Cesarman, 2013; Said et al., 1996; Soulier et al., 1995). The narrow host
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range of EBV and KSHV makes it difficult to study molecular mechanisms of their lytic
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replication and latency in experimental animals.
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Murine gammaherpesvirus (MHV) 68, isolated from field mice such as bank
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voles and wood mice (Blaskovic et al., 1980), is a member of 2-herpesviruses (or
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Rhadinovirus) and shares sequence homology with the human gammaherpesviruses
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(Efstathiou et al., 1990; Virgin et al., 1997). Because of the biological and genetic
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similarity, MHV68 infection is considered to be a useful animal model of human
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gammaherpesviruses (Barton et al., 2011; Simas & Efstathiou, 1998). In addition,
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MHV68 lytically infects cells of various species (Svobodova et al., 1982), allowing us to
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study the lytic infection of gammaherpesviruses.
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MHV68 has at least 80 genes (Virgin et al., 1997), and shares many genes
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involved in DNA replication and virion assembly with other herpesviruses (Dunn et al.,
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2003; Virgin et al., 1997; Yu et al., 2003). The open reading frame (ORF) 35 is
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conserved among gammaherpesviruses including KSHV (Russo et al., 1996), herpesvirus
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saimili (Albrecht et al., 1992), EBV (Baer et al., 1984), and equine herpesvirus 2 (Telford
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et al., 1995). The ORF35 gene has been shown to be essential for MHV68 lytic infection,
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by a signature-tagged mutagenesis screening (Song et al., 2005), but its function in
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MHV68 replication has not been determined.
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Although KSHV ORF35 protein is predominantly localized in the cytoplasm
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(Masa et al., 2008), its role during lytic infection is also poorly understood. In herpes
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simplex virus (HSV)-1 and -2, UL14, a homologue of ORF35 (Dolan et al., 1998;
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McGeoch et al., 1988; Mills et al., 2003), exists as a minor component of the virion
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particle (Cunningham et al., 2000; Wada et al., 1999) and exerts multiple functions
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including apoptosis inhibition (Yamauchi et al., 2003) and chaperone activity (Yamauchi
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et al., 2002). An HSV-1 UL14 mutant virus exhibits lower growth efficiency in vitro and
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lower pathogenicity in experimental mice (Cunningham et al., 2000).
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In this study, we characterized the properties of MHV68 ORF35. Our results
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show that ORF35 is not essential for MHV68 replication but plays an important role after
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the expression of late viral proteins. Intranasal infection of mice with MHV68 lacking
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ORF35 resulted in lower virus yield during acute replication in the lung. Further, the
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mutant virus had a defect in reactivation from infected splenocytes.
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RESULTS
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ORF35 is a phosphorylated protein.
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To characterize the ORF35 protein, we prepared a plasmid expressing ORF35 tagged
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with the Flag epitope at the N-terminus. Immunoblotting analysis with anti-Flag antibody
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revealed two specific bands at 26 kD and 28 kD in transfected HEK293T cells (Fig. 1a).
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We then examined three ORF35 mutants in which 25, 32, or 55 residues from the
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C-terminus were truncated (C25, C32 and C55) (Fig. 1b and 1c). C25 exhibited two
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bands like the full-length ORF35, while only a single band was detected in C32- or
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C55-expressing cells (Fig. 1c). MHV68 ORF35 includes 11 serine and 4 threonine
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residues which are predicted to be phosphorylated when analyzed by NetPhos 2.0 Server
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(Center for Biological Sequence Analysis, Technical University of Denmark
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[http://www.cbs.dtu.dk/services/NetPhos/]) (Fig. 1b). As both C32 and C55 lacked
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three serine residues at positions 124, 126, and 129 which were phosphorylation
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candidates (Fig. 1b), we hypothesized that the upper band of full-length ORF35 could be
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caused by phosphorylation at these serine residues. We constructed the AAA mutant that
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has all of the three serine residues replaced with alanine. As expected, the AAA mutant
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gave a single band with the similar molecular weight to that of the lower band of the
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full-length ORF35 (Fig. 1d), indicating that the upper band is a phosphorylated ORF35 at
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these serine residues. We evaluated which serine residues of the three were important for
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the phosphorylation, but the results indicated that all of the three serine residues influence
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the phosphorylation status to some extent (data not shown). To examine the involvement
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of other residues in the phosphorylation, we treated the full-length ORF35 or the AAA
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mutant with alkaline phosphatase. The treatment diminished the upper band of the
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full-length ORF35 (Fig. 1d). However, the phosphatase did not affect the mobility of the
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26kD bands of the full-length ORF35 and the AAA mutant, indicating that the three
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serine residues are solely responsible for the phosphorylation of ORF35.
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A putative coiled-coil motif in ORF35 mediates homo-oligomerization.
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MHV68 ORF35 protein is predicted to have two coiled-coil motifs (amino acid positions
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41-73 and 100-112) according to ISREC-Server (SIB ExPASy Bioformatics Resources
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Portal [http://embnet.vital-it.ch/software/TMPRED_form.html]) (Fig. 2a). As the motif is
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known to mediate protein-protein interactions, we investigated the oligomer formation of
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the ORF35 protein. Co-immunoprecipitation (Co-IP) assay using Flag- and HA-tagged
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ORF35 proteins revealed the interaction between ORF35 proteins (Fig. 2b lane 2 and Fig.
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2c lane 2). To determine the region(s) responsible for the interaction, we prepared a series
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of the N- or C-terminus truncated mutants (N17, N26, N33, N56, N76, C16,
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C25, C32, and C55), and investigated the interaction between the full-length ORF35
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and each of the truncation mutants or the AAA mutant. The results showed that the
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residues at positions 34 to 76 were critical, but the phosphorylation was dispensable, for
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the interaction (Fig. 2b and 2c). Similar results were obtained by Blue-Native (BN)
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polyacrylamide gel electrophoresis (PAGE). The full length ORF35 and N17, N26 and
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N33 mutants exhibited two bands around 18 kD and 66 kD likely corresponding to
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monomer and multimer forms, respectively (Fig. 2d). N56 had the diminished band
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around 66 kD and formed much smaller complexes. Truncation of N-terminus 76
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residues almost abolished the complex formation (Fig. 2d). The data implicate the first
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predicted coiled-coil motif at positions 41 to 73 in oligomer formation of ORF35.
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Interestingly, the C-terminus truncation mutants tended to efficiently make complexes for
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unknown reasons (Fig. 2c).
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Repeats of 7 amino acid residues, usually expressed as (a-b-c-d-e-f-g)n , are required to
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form the coiled-coil motif. In general, nonpolar residues at positions a and d facilitate
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hydrophobic interactions, whereas polar residues at positions e and g provide binding
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specificity through electrostatic interactions (Fig. 2a). We analyzed the importance of the
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charged residues at positions e and g for oligomerization using three substitution mutants;
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K44E/E49R, R56E/H58E/E63R, and K105E/E107R (Fig.2a). By Co-IP assay, we found
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that the K44E/E49R and R56E/H58E/E63R exhibited reduced ability to oligemerize with
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the full-length ORF35, whereas the K105E/E107R still interacted efficiently (Fig. 2c).
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Taken together, the charged amino acid residues in the first putative coiled-coil motif are
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likely critical for the homo-oligomerization of the ORF35 protein.
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ORF35 is mainly localized in the cytoplasm.
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Next, we performed immunofluorescence staining to investigate the subcellular
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localization of ORF35. When expressed by plasmid-mediated transfection, Flag-ORF35
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was localized predominantly in the cytoplasm, and only a small part was detected in the
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nucleus (Fig. 3a and 3b). ORF35-HA and the AAA mutant were also similarly distributed
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(data not shown). This pattern of the subcellular distribution is similar to that of KSHV
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ORF35 in transfected cells (Masa et al., 2008). Further, the localization of Flag-ORF35
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expressed by transfection was not affected by MHV68 infection (Fig. 3b and 3c).
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ORF35 is required for efficient replication in vitro.
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To examine the role of ORF35 in the life cycle of MHV68, we constructed a bacterial
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artificial chromosome (BAC) clone encoding the ORF35 stop mutant (35S) virus, which
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possesses an ectopic stop codon, a frameshift mutation, and a SacI recognition site after
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the overlapping sequence with ORF34 (Fig. S1a). We also produced a BAC clone to
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generate the recombinant virus expressing unphosphorylated ORF35 (AAA virus) (Fig.
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1d). Restriction enzyme digestion and agarose gel electrophoresis confirmed the expected
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structures for these BAC-cloned genomes (Fig. S1b).
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ORF35 has been reported to be an essential gene (Song et al., 2005). However, NIH3T3
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cells transfected with BAC-35S developed cytopathic effect (CPE) (data not shown),
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suggesting that the gene may not be an essential gene. There was no unexpected mutation
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in the targeted region of the virus genome in the infected cells (data not shown). We also
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excluded the contamination of 35S with the WT virus, by SacI digestion (Fig.S1c). The
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SacI marker-reverted (35R) virus and the AAA virus were also recovered without
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difficulty. After removal of the BAC cassette, all these viruses were used for further
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experiments.
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Next, we analyzed the growth of the recombinant viruses. The titers of the cell-associated
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and cell-free 35S virus were around 20-30 times lower than those of WT virus at later
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time points regardless of M.O.I. (Fig. 4a and 4b). On the other hand, the yield of the 35S
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virus in NIH3T3/F35 cells stably expressing Flag-ORF35 (Fig S2) was several times
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higher than that in parental NIH3T3 cells (Fig. 4c), indicating that ORF35 is indeed
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required for efficient virus growth. Further, the 35R virus exhibited growth kinetics
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closely similar to WT virus, ascertaining that the attenuated growth of the 35S virus was
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not due to unexpected mutations in other genes. The phosphorylation of ORF35 appears
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to exhibit no effect on virus growth, as the AAA virus grew as efficiently as WT and 35R
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viruses (Fig. 4a and 4b). Taken together, the data indicate that ORF35 is necessary for
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efficient virus growth.
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The 35S virus exhibits normal genome replication and lytic protein expression.
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To determine which step(s) in replication of 35S virus was disturbed, we analyzed the
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genome replication in virus-infected cells. In WT virus-infected cells, the amount of the
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viral genome at 24 h after infection was around ten times higher than that at 3 h after
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infection (Fig. 5a). When infected cells were treated with phosphonoacetic acid (PAA), a
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viral DNA polymerase inhibitor, no genome replication was observed, as expected. All of
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35S, 35R, AAA and WT viruses exhibited similar levels of the genome amplification
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(Fig. 5a), indicating that ORF35 does not function before the DNA replication step.
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Next, the expression of virus proteins in infected cells was also analyzed. In WT
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virus-infected cells, many peptide bands were detected with anti-MHV68 serum at 24 h
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after infection, and most of them disappeared when the cells were treated with PAA (Fig.
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5b). Thus, most of the signals seen in WT virus-infected cells were likely viral late
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proteins, as the expression of these proteins is known to be dependent on viral DNA
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replication (Johnson & Everett, 1986; Mavromara-Nazos & Roizman, 1987). In 35S
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virus- or AAA virus-infected cells, the pattern and intensity of the bands detected were
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almost identical to those in WT virus-infected cells. The data indicate that the lack of
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ORF35 does not affect the expression of late proteins.
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ORF35 is needed for efficient lytic replication and latency in mice.
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To examine the growth of recombinant viruses in vivo, anesthetized C57BL/6 mice were
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intranasally infected with them, and virus titers in the lung were determined at 3 and 6
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days post-infection. At each time point, the titer of the 35S virus was more than hundred
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times lower than that of the WT virus, whereas 35R and AAA viruses grew comparably
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to the WT virus (Fig.6a). The data indicate that ORF35 plays an important role in
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efficient virus production in vivo. Again, there was no apparent effect of the ORF35
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phosphorylation on lytic replication in vivo.
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Next, we examined infected mice at the latent phase of infection. Splenomegaly as
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evaluated by spleen weight, viral genomic copy number in splenocytes, and the frequency
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of ex vivo reactivation from splenocytes were analyzed at 17 and 24 days after infection.
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The mean spleen weight of 35S virus-infected mice was lower than that of WT, 35R, or
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AAA virus-infected mice at 17 and 24 days after intranasal infection (though not
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statistically significant at the latter time point), possibly reflecting reduced growth of the
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35S virus in the lung. By contrast, the difference in spleen weight was not observed
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among mice infected intraperitoneally with these 4 types of viruses (Fig. 6b). The mean
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genome copy number of 35S virus-infected mice was significantly reduced as compared
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with that of mice infected with the other types of viruses after intranasal infection (Fig.
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6c). Again, the difference was not observed when mice were infected intraperitoneally
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with these viruses. The data indicate that the 35S virus is able to establish latent infection
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in the spleen almost as efficiently as the WT virus when inoculated intraperitoneally.
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Notably, the reactivation frequency of the 35S virus was below the detection limit
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independent of the route of infection (Fig. 6d), indicating that the 35S virus could not be
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reactivated from the latent state. As preformed infectious viruses in disrupted splenocytes
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were below the detection limit for any mouse group regardless of the infection route (data
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not shown), the CPEs observed were caused not by preformed viruses, but by reactivation.
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Taking together, we conclude that ORF35 also influences reactivation of MHV68 from
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latency.
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DISCUSSION
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MHV68 ORF35 has been shown to be essential for lytic infection (Song et al., 2005), but
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our data using the 35S virus indicate that the gene is not essential but necessary for
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efficient lytic infection in vitro and in vivo. What accounts for the discrepancy? ORF35
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homologues are located in the gene cluster comprising ORF34 to ORF38 (Baer et al.,
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1984; Russo et al., 1996; Virgin et al., 1997). In KSHV-infected cells, two polycistronic
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transcripts are synthesized from the gene cluster, the 3.4 kb mRNA encoding ORF35 to
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ORF37 and 4.2 kb mRNA encoding ORF34 to ORF37 (Haque et al., 2006; Masa et al.,
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2008). As MHV68 ORF35 is also encoded in a similar manner (data not shown), the
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insertion of transposons into ORF35 may have affected the stability of the polycistronic
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mRNA, thereby affecting the expression of the genes (ORF35 to ORF38) encoded in the
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transcript. Although ORF36, ORF37 and ORF38 were shown to be nonessential
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(knockout of ORF36 resulted in an attenuated phenotype) (Song et al., 2005), the
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combined defect of ORF35 to ORF38 may have been lethal for MHV68. Alternatively, a
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transcriptional control region for an essential gene(s) nearby such as ORF34 may be
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present in the ORF35 coding region.
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Since the genome replication and expression of lytic proteins in 35S virus-infected cells
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were comparable to those in WT and 35R virus-infected cells, ORF35 likely functions in
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the assembly and/or egress step after the production of late proteins. Our finding is
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consistent with the previous report that HSV-1 UL14, a homologue of ORF35 (Mills et
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al., 2003), is necessary for the efficient nucleocapsid egress from the nucleus
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(Cunningham et al., 2000). It is estimated that HSV 1 UL14 is present at no more than a
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few dozen molecules per virion (Cunningham et al., 2000). ORF35 homologues have not
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been detected in virions of MHV68, KSHV, and EBV (Bortz et al., 2003; Johannsen et
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al., 2004; Vidick et al., 2013; Zhu et al., 2005) although ORF35 of rhesus monkey
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rhadinovirus was found in the virion (O'Connor & Kedes, 2006).
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The 35S virus was able to replicate lytically in NIH3T3 cells in vitro and the mouse lung
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in vivo (albeit at lower efficiencies) and exhibited virus genomic load similar to those of
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the other recombinant viruses in splenocytes after intraperitoneal infection, but it failed to
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reactivate from splenocytes. Since ORF35 is not detected in latently infected cells
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(Ebrahimi et al., 2003; Martinez-Guzman et al., 2003), it may function at a step(s) after
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reactivation triggering. The MHV68 lytic replication in myeloma cells was reported to be
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inefficient (Forrest & Speck, 2008; Sunil-Chandra et al., 1993). This low permissivity of
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B cells to MHV68 may be a reason for the inability of the 35S virus to reactivate from
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latently infected cells, because even mild growth impairment of the 35S virus, as
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compared with the WT virus, would become highly significant in this context.
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Structural analysis revealed that there are two putative coiled-coil motifs in ORF35. The
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coiled-coil motif is known to mediate protein-protein interactions for such functions as
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transcription, intracellular transport, and virus entry (Mason & Arndt, 2004; Wang et al.,
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2012). Charged amino acid residues at positions e and g in the coiled-coil motif usually
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determine the binding specificity for target proteins, and recruit viral or host proteins by
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making complexes. The first motif was found to be involved in homo-oligomerization of
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ORF35. The second motif may also have some roles in the function of ORF35. To
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explore this possibility, we are currently studying host proteins interacting with ORF35.
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MATERIALS AND METHODS
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Plasmid constructions. The putative ORF35 coding region (Virgin et al., 1997) was
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tagged with the Flag-epitope at the N-terminus, and subcloned into an expression vector
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pCA7, a derivative of pCAGGS (Niwa et al., 1991), or a retrovirus vector pMXs-IP
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(Kitamura et al., 2003). ORF35 tagged with the influenza hemagglutinin (HA) epitope at
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the C-terminus was subcloned into pCA7. All truncation and amino acid substitution
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mutants were generated by PCR-based mutagenesis, tagged with the Flag epitope, and
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subcloned into pCA7. Primer sequences used for subcloning and mutagenesis will be
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provided upon request. The coding sequence of the P1 bacteriophage cre-recombinase
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was subcloned into pMXs-IP.
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Cells. HEK293T and NIH3T3 cells were maintained in Dulbecco’s Modified Medium
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supplemented with 10% FBS and Penicillin/Streptomycin referred to here as culture
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medium (CM). Plat-E cells, a retrovirus packaging cell line (Morita et al., 2000), were
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maintained in CM supplemented with 1 g Puromycin ml-1 (InvivoGen) and 10 g
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Blasticidin ml-1 (InvivoGen). NIH3T3 cells stably expressing Flag-tagged ORF35
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(NIH3T3/F35 cells) and cre-recombinase (NIH3T3/Cre cells) were generated by
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infecting the cells with respective retroviruses generated form Plat-E cells transfected
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with appropriate retrovirus plasmids, followed by 1 g Puromycin ml-1 selection and
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limiting dilution.
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Viruses. To prepare virus stocks, NIH3T3 cells were infected with each of the
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recombinant viruses, and both cells and culture medium were collected when CPE was
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complete. The harvested samples were centrifuged, and the pellets were suspended in CM.
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After freezing and thawing twice, the samples were centrifuged and the supernatant was
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kept at -80C. Infectious viruses were titrated by limiting dilution assay using NIH3T3
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cells and expressed as TCID50. Virus titers of the 35S virus stock were comparable when
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examined by limiting dilution assay using NIH3T3 and NIH3T3/F35 cells.
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Antibodies and antiserum. Anti-MHV68 sera were harvested from three
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MHV68-infected mice and pooled. Rabbit anti-Flag polyclonal antibody (pAb) (Sigma;
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F7425), mouse anti-Flag monoclonal antibody (mAb) (Sigma; F1804), Rabbit anti-HA
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pAb (MBL; 561 ), rabbit anti-Lamin A pAb (Santa Cruz Biotech; sc-20680), mouse
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anti-Tubulin mAb (Santa Cruz Biotech; sc-5286), goat anti-rabbit IgG conjugated with
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horse radish peroxidase (HRP) (Amersham Biosciences), goat-anti mouse IgG conjugated
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with HRP (Jackson ImmunoResearch), donkey anti-mouse IgG (H+L) conjugated with
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Alexa Fluor 488 or 594 (Molecular Probes), and donkey anti-rabbit IgG (H+L)
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conjugated with Alexa Fluor 488 or 594 (Molecular Probes) were purchased from
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indicated suppliers.
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Immunoprecipitaition. HEK293T cells were transfected with appropriate combinations
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of expression plasmids. At 24 h after transfection, the cells were washed with PBS and
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lysed with IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM
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EDTA, 5% glycerol) supplemented with the protease inhibitor cocktail (Sigma).
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Insoluble debris was removed by centrifugation, and a small amount of the supernatant
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was kept as the whole cell lysate (WCL) sample. The remaining supernatant was
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pre-cleared by incubating with Protein-A Sepharose (GE Healthcare), and then mixed
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with fresh Protein-A Sepharose and appropriate antibody. After washing intensively with
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IP buffer without the protease inhibitor cocktail, proteins in the precipitate were
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solubilized in the SDS loading buffer (Ito et al., 2013) and boiled.
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Dephosphorylation assay. Dephosphorylation assay was performed as previously
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reported (Jia et al., 2005). HEK293T cells were transfected with the expression plasmid
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of interest. At 48 h after transfection, the cells were re-suspended into NEB Buffer 3 (100
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mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT; New England BioLabs)
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followed by freezing and thawing twice. Cell debris was removed by centrifugation and
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the supernatant was treated with calf intestine alkaline phosphatase (New England
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BioLabs) or mock treated. After adding the same volume of 2SDS loading buffer, the
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sample was boiled.
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Immunoblotting. NIH3T3 cells were infected with each of the recombinant viruses at an
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M.O.I. of 0.5. Proteins in WCL at 24 h after infection were separated in
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SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride (PVDF)
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membranes. The membrane was blocked with Tris-buffered saline containing 0.05%
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Tween-20 (TBS-T) and 5% skim milk, followed by the treatment with suitable
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combinations of antibodies. After washing three times, the membrane was treated with
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Chemi-Lumi One Super (Nacalai Tesque) for signal detection using VersaDoc 5000
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imager (Bio-Rad).
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BN- PAGE. HEK293T cells were transfected with appropriate expression plasmids. At
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24 h after transfection, the cells were washed with PBS and lysed with NativePAGE
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Sample Buffer (Invitrogen) supplemented with 5% n-dodecyl β-D-maltoside (DDM;
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Invitrogen) and 0.05% Coomassie brilliant blue G250 (Invitrogen). Lysed proteins were
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separated in 5-20% gradient gel, incubated with 20mM Tris-HCl (pH7.4) containing
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150mM Glycine and 0.1% SDS at room temperature for 5 min, and then transferred onto
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PVDF membranes. The membranes were incubated with 50mM Tris-HCl (pH7.4)
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containing 2% SDS and 0.8% 2-mercaptoethanol at 55C for 30 min, and followed by
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blocking and immunodetection as above.
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Indirect immunofluorescent assay. NIH3T3 cells were infected with MHV68 for 1 h or
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left uninfected. The supernatant was replaced with fresh medium and the cells were
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transfected with pCA7-Flag-ORF35. At 17 h after transfection, the cells were stained
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with appropriate combinations of antibodies (Ito et al., 2013). The stained cells were
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observed under a confocal microscope (Radiance 2100; Bio-Rad).
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Virus growth in cultured cells. NIH3T3 and NIH3T3/F35 cells were infected with each
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of the recombinant viruses for 1 h at an M.O.I. of 0.05 or 3. At indicated days after
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infection, infectious viruses in cells and supernatants were titrated, respectively.
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Animal infection. All animal experiments were reviewed by the Institutional Committee
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of Ethics on Animal Experiments of the Faculty of Medicine, Kyushu University, and the
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facility guidelines for animal experiments and care were strictly followed. Sex- and
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age-matched C57BL/6 mice were anesthetized by the mixture of medetomidine,
381
midazolam, and butorphanol, and then infected intranasally or intraperitoneally with 105
382
TCID50 of the recombinant viruses. The infected mice were housed in ventilated isolation
383
cages during infection. At 3 and 6 days after infection, virus titers in lung homogenates
384
were determined (Flach et al., 2009). At 17 and 24 days after infection, spleens were
385
harvested, weighed, and prepared for single cell suspensions. After removing red blood
386
cells, splenocytes were counted and used for the determination of reactivation frequency
387
and DNA isolation.
388
389
Ex vivo reactivation assay. A series of three-fold dilutions of splenocytes mixture
390
starting from 1.5  105 cells / well were overlaid on NIH3T3 cells seeded on 96-well
391
plate beforehand (Adler et al., 2001; Weck et al., 1996). The appearance of CPE in each
392
well was observed up to 2 weeks after overlay and the reactivation frequency of each
393
diluent was calculated. The presence of pre-formed infectious virus was checked
394
following the same procedure using splenocytes disrupted by freezing and thawing twice.
395
396
Measurement of viral genome copy number by real-time PCR. NIH3T3 cells infected
397
with each of the recombinant viruses were cultured with or without 200 g PAA (Sigma)
398
ml-1. DNA was extracted from virus-infected NIH3T3 cells at 3 h and 24 h after infection,
399
or mouse splenocytes using the GenElute Mammalian Genomic DNA Miniprep Kit
400
(Sigma) following the manufacturer's instructions. Quantitative PCR was carried out
401
using SYBR Premix Ex Taq II (Takara) and a Light Cycler 1.5 (Roche). A primer set for
402
ORF57 was used to quantify the virus genome and mouse ribosomal L8 gene (L8) was
403
also amplified for normalization (TABLE 1) (Flach et al., 2009). The PCR fragments of
404
ORF57 and L8 were subcloned and used as standard plasmids. For the copy number in
405
virus-infected NIH3T3 cells, data are shown as the copy number at 24 h after infection
406
relative to that at 3 h after infection. For the genomic load in splenocytes, the data are
407
shown as the copy number of ORF57 relative to 1000 copies of the ribosomal L8 gene.
408
409
Statistics. Statistical significance was analyzed using the unpaired Student’s t-test.
410
411
ACKNOWLEDGMENTS
412
We appreciate the technical assistance from The Research Support Center, Kyushu
413
University Graduate School of Medical Sciences. We also thank H. Adler and T.
414
Kitamura for generously providing the MHV68 BAC plasmid, and the retrovirus vector
415
and packaging cells, respectively. This study was supported in part by JSPS KAKENHI
416
grant 26460558.
417
418
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419
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623
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TABLE
TABLE. 1 Primer sequence used for quantitative PCR
Name
ORF57-upper
ORF57-lower
L8-upper
L8-lower
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
5' 
CCTGAAGAGAACCCCTTGC
CGCCTGGCTGATACTTTAGG
CATCCCTTTGGAGGTGGTA
CATCTCTTCGGATGGTGGA
3'
644
FIGURE LEGENDS
645
Fig. 1 ORF35 is a phosphorylated protein. (a) Expression of Flag-ORF35 protein.
646
HEK293T cells were transfected with the vector expressing Flag-ORF35 or empty
647
plasmid. Whole cell lysates at 24 h after transfection were immunoblotted using anti-Flag
648
pAb. The addition of the Flag epitope reduced the protein mobility in the SDS-PAGE.
649
(b) Putative phosphorylated residues in MHV68 ORF35. Amino acid sequence of ORF35
650
is shown with single letters. Putatively phosphorylated serine (S) and threonine (T)
651
residues are highlighted. Underlines indicate the deleted region of the truncation mutants.
652
aa; amino acid residues. (c) Expression of truncated ORF35 proteins. HEK293T cells
653
were transfected with each of expression vectors, and the expression of C-terminally
654
truncated ORF35 proteins tagged with Flag epitope was analyzed by immunoblotting
655
using anti-Flag pAb. (d) Dephosphorylation assay. HEK293T cells were transfected with
656
each of expression plasmids, and cell lysates were treated with calf intestine alkaline
657
phosphatase (ALP) or mock treated. The samples were analyzed by immunoblotting.
658
659
Fig. 2 A putative coiled-coil motif in ORF35 mediates homo-oligomerization. (a) A
660
schematic illustration of truncation mutants and the predicted coiled-coil motifs in
661
ORF35. ORF35 protein and Flag epitope are shown as white and dark-gray boxes,
662
respectively. The numbers indicate amino acid residue positions of ORF35 in the
663
full-length and mutant constructs. Amino acid sequences of predicted coiled-coil motifs
664
in ORF35 (positions 41 to 73 and 100 to 112) are shown with single capital letters. Small
665
letters above the residues indicate the tentative positions (a to g) in the coiled-coil motif.
666
Amino acid residues replaced in the mutants are highlighted. (b and c) Co-IP assay of
667
N-terminally truncated mutants and AAA mutant (b) and C-terminally truncated mutants
668
and mutants with amino acid substitutions (c). HEK293T cells were transfected with each
669
of expression plasmids along with the ORF35-HA expression vector. At 24 h after
670
transfection, proteins were precipitated with anti-Flag mAb and examined by
671
immunoblotting. (d) BN-PAGE analysis. HEK293T cells were transfected with each
672
expression plasmid. At 24 h after transfection, proteins were separated by BN-PAGE,
673
blotted onto PVDF membrane, and reacted with anti-Flag mAb.
674
675
Fig. 3 Subcellular localization of ORF35 protein. (a and b) NIH3T3 cells were
676
transfected with pCA7-Flag-ORF35. At 24 h after transfection, the Flag epitope (red) and
677
Tubulin (a) or Lamin A (b) (green) were doubly stained. (c) NIH3T3 cells were infected
678
with MHV68 at an M.O.I. of 3 for 1 h and then transfected with pCA7-Flag-ORF35. At
679
17 h after transfection, the Flag epitope (red) and Lamin A (green) were doubly stained.
680
681
Fig. 4 Growth kinetics of ORF35 mutant viruses in vitro. (a and b) Growth kinetics of
682
recombinant viruses. NIH3T3 cells were infected with each of the BAC-removed viruses
683
at an M.O.I. of 0.05 (a) or 3 (b), and infectious viruses in culture supernatant or
684
cell-associated virus were titrated at indicated days after infection. (c) Growth kinetics of
685
the 35S virus in NIH3T3 and NIH3T3/F35 cells. Respective cells were infected with the
686
35S virus at an M.O.I. of 0.05, and infectious viruses were titrated at indicated days after
687
infection.
688
689
Fig. 5 Genome replication and lytic protein expression in virus-infected cells. (a)
690
Genome replication in NIH3T3 cells. Cells were infected with indicated viruses at an
691
M.O.I. of 0.5 and incubated with or without PAA. At 3 h and 24 h after infection, DNAs
692
were isolated and analyzed by real-time PCR. The data were normalized by the cellular
693
ribosomal L8 gene and expressed as relative increase of the copy number at 24 h after
694
infection as compared with that at 3 h. (b) Expression of lytic proteins. NIH3T3 cells
695
were infected with indicated viruses at an M.O.I. of 0.5 and incubated with or without
696
PAA. Expression of lytic proteins was analyzed by immunoblotting using anti-MHV68
697
serum.
698
699
Fig. 6 Lytic replication and latency of recombinant viruses in mice. (a) Lytic
700
replication in the lung. C57BL/6 mice were infected intranasally with 105 TCID50 of
701
indicated viruses. At 3 and 6 days after infection, virus titers in the lung were determined.
702
Each symbol represents an individual mouse and the bar indicates the mean value. N; the
703
number of mice in each group. (b) Evaluation of splenomegaly at 17 and 24 days after
704
infection. Mean spleen weight with standard deviation is shown. N; the number of mice
705
in each group. (c) Viral genomic load in the spleen at 17 and 24 days after infection. The
706
copy number of ORF57 relative to 1000 copies of the ribosomal L8 gene is shown. Each
707
symbol represents an individual mouse and the bar indicates the mean value. N; the
708
number of mice in each group. (d) Ex vivo reactivation from splenocytes. Mice were
709
infected with 105 TCID50 of indicated viruses intranasally or intraperitoneally. At 17 and
710
24 days after infection, a set of three fold dilutions of splenocyte mixture were overlaid
711
onto NIH3T3 cells. Frequency of CPE positive well was calculated. N; the number of
712
mice analyzed in each group.
713
714