Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Slot 511, 4301 W. Markham Street, Little Rock, AR 72205, USA1
Author for correspondence: Wayne Gray. Fax +1 501 686 5359. e-mail graywaynel{at}exchange.uams.edu
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Abstract |
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Introduction |
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Little is known about the molecular biology of CCV or the virus replication cycle. The CCV genome (134·2 kb) contains two identical direct repeat regions (18·5 kb each) that flank the 97·1 kb unique region (Fig. 1) (Davison, 1992
). Each direct repeat contains 14 open reading frames (ORFs), while the unique region contains 65 ORFs, for a total of 79 distinct ORFs in the genome (Davison, 1992
). Although CCV is not closely related to any other known herpesvirus, some CCV genes share limited homology with salmonid herpesvirus genes (Davison, 1998
).
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An understanding of CCV gene expression is fundamental to determine the role of CCV genes in virus replication, pathogenesis and latency. The repeat regions of herpesvirus genomes often contain genes that encode regulatory proteins as well as gene products which are important in virus pathogenesis, including latency associated transcripts (Baxi et al., 1995 ; Mitchell et al., 1990
; Priola & Stevens, 1991
). In this study, we have identified the transcripts encoded by the CCV direct repeat region(s) and characterized the transcriptional regulation of the CCV direct repeat genes.
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Methods |
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Protein synthesis inhibition.
CCO monolayers were incubated in methionine deficient media with or without cycloheximide (CH; 100 µg/ml) for 1 h. Cellular proteins were then labelled in vivo with 10 µCi/ml [35S]methionine. At the same time-point, cells were infected at an m.o.i. of 4 p.f.u. per cell (or mock-infected). At 1, 1·5, 3 and 6 h post-infection (p.i.), cells were harvested and washed in PBS. Cells were pelleted, resuspended in solubilization buffer (25 mM TrisHCl, 250 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0·5% deoxycholate) and stored at -70 °C. Samples were sonicated on ice and cellular debris was pelleted. Supernatants were precipitated on ice with BSA and trichloroacetic acid (TCA) for 15 min. The precipitates were collected on Whatman GF/C glass-fibre discs, washed with ice cold 10% TCA and then with ethanol. The filters were dried and radioactivity was measured in a liquid scintillation counter. The radioactivity (c.p.m.) of samples that were not treated with CH represented 100% protein synthesis under each set of conditions.
CCV DNA synthesis inhibition.
CCO cells were infected with 4 p.f.u. CCV per cell or mock-infected in the presence (or absence) of cytosine -D-arabinofuranoside (Ara-C; 50 or 100 µg/ml). At 1 and 6 h p.i., cells were removed and washed in PBS. Total cell DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). DNA from each sample (6 µg) was denatured in 0·25 M NaOH, 0·5 M NaCl; diluted in 0·1x SSC (1x SSC=150 mM NaCl, 15 mM sodium citrate), 0·125 M NaOH; and applied to a positively charged nylon membrane (Boehringer Mannheim). The resulting dot blot was neutralized in 1·5 M NaCl, 0·5 M TrisHCl (pH 7·4) and cross-linked by exposure to ultraviolet light. Prior to hybridization, the blot was denatured in 0·4 M NaOH, soaked in 0·2 M TrisHCl (pH 7·5) and incubated in prehybridization solution (10% dextran sulfate, 1% SDS, 1 M NaCl). A digoxigenin (DIG)-labelled CCV double-stranded DNA probe (ORF 59, nucleotide 78703 to 79740 on the CCV genome) was denatured and added directly to the prehybridization solution for hybridization at 65 °C for 1618 h. Alkaline phosphatase-conjugated anti-DIG antibody and the chemiluminescent substrate disodium 3-(4-methoxyspiro (1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1]decan)-4-yl)phenyl phosphate (CSPD; Boehringer Mannheim) were used for detection of the probe by exposure to film. The relative signal from each dot was measured densitometrically and used to calculate the relative percentage of DNA synthesis in each sample.
RNA isolation and Northern hybridization.
CCO cells were infected with 4 p.f.u. of CCV per cell. For CCV IE RNA, the cells were incubated in 100 µg/ml CH for 30 min prior to infection as well as during infection. CCV IE RNA was isolated at 1 and 1·5 h p.i. For CCV early RNA, cells were incubated in 100 µg/ml Ara-C during infection and total RNA was isolated at 6 h p.i. For late CCV RNA, cells were incubated in media without inhibitors and total RNA was isolated at 6 h p.i. RNA was also isolated from CCO cells in the absence of CCV infection (mock-infected). Total cell RNA was isolated from the cells using the RNAzol B reagent (Tel Test I), an adaptation of the guanidinium thiocyanate method (Chomczynski & Sacchi, 1987 ). RNA was quantified by absorbance measurements and stored in DEPC-treated water at -70 °C until used.
For Northern hybridization analysis RNA (10 µg) samples were treated with dimethyl sulfoxide and deionized glyoxal. Samples (7 µg) were fractionated by electrophoresis in a 1·2% agarose gel containing 10 mM sodium phosphate (pH 7), 10 mM iodoacetic acid and 1µg/ml ethidium bromide. Ribosomal RNA (28S and 18S) was visualized under ultraviolet light to ensure equivalent amounts of RNA in each lane. The RNA was transferred to a positively charged nylon membrane (Boehringer Mannheim). Blots were prehybridized in 10% dextran sulfate, 1% SDS, 1 M NaCl. Portions of CCV genes were amplified by PCR and then labelled with DIG. The CCV-specific double-stranded DNA probes were added directly to the prehybridization solution for hybridization at 65 °C for 1618 h. CCV transcripts were detected by chemiluminescence and film exposure employing alkaline phosphatase-conjugated anti-DIG antibody and CSPD.
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Results |
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The inhibition of viral DNA synthesis by Ara-C was measured in CCV-infected (and mock-infected) CCO cells by DNA hybridization (Fig. 2). A DNA probe specific for CCV ORF 59 was used to detect CCV DNA. No specific signal was detected in mock-infected CCO DNA samples. Only the input level of CCV DNA was detected in total cell DNA isolated at 1 h p.i. from CCV-infected cells in the presence or absence of Ara-C. At 6 h p.i. a 2·2-fold increase in the amount of CCV DNA was detected in the absence of Ara-C, indicating that viral DNA replication had occurred. In the presence of 50 µg/ml Ara-C the synthesis of CCV DNA was inhibited by 50%, and in the presence of 100 µg/ml Ara-C the synthesis of CCV DNA was inhibited by 90%.
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Transcriptional regulation of CCV genes
Northern analysis of CCV RNA indicated that ORFs 1 and 3 were the only IE genes within the CCV direct repeat region (Fig. 3); Table 1
. At both 1 and 1·5 h p.i., transcription of the 3·0 kb ORF 1 transcript (IE 1) and the 1·6 kb ORF 3 transcript (IE 3) was not inhibited by CH. The 3·0 kb IE 1 transcript remained at high levels at 6 h p.i. However, the abundance of the 1·6 kb IE 3 transcript was significantly reduced at 6 h p.i.
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Time-course of CCV gene transcription.
The progression of CCV transcription in infected CCO cells was examined by Northern blot hybridization analysis employing specific CCV gene probes (Table 1). Total RNA was isolated from infected cells at hourly intervals from 1 to 6 h p.i. The time-course of transcripts encoded by ORFs 1, 2, 3, 5, 14 and 59 is shown in Fig. 5
. The 3·0 kb IE 1 and 1·6 kb IE 3 transcripts were readily detected as early as 1 h p.i. The IE 1 transcript was highly expressed throughout the 6 h infection. The IE 3 transcript was detected at 1 to 4 h p.i., but was more difficult to detect at 5 and 6 h p.i. This result is consistent with a previous finding that the ORF 3 IE transcript has a short half-life (Silverstein et al., 1998
). The CCV early transcripts (2·9 kb ORF 2, 2·9 kb ORF 3, 1·4 kb ORF 5 and 1·4 kb ORF 14 transcripts) were detected in relatively high abundance from 2 to 6 h p.i. In general, early transcription peaked around 3 to 4 h p.i. The 1·2 kb ORF 59 late transcript was not readily detected until 4 h p.i., after the onset of viral DNA synthesis.
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Discussion |
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Metabolic inhibitors that are commonly used in studying the transcriptional regulation of mammalian herpesviruses were effective in CCV-infected CCO cells. Ara-C inhibited 90% of CCV DNA synthesis in these cells and effectively blocked the production of late CCV transcripts. In addition, CH blocked greater than 97% of protein synthesis in these cells for up to 6 h p.i. However, our analysis, as well as that of Silverstein et al. (1998) , suggests that the CH block leaks over time to allow transcription of some CCV genes that are not IE. We defined CCV IE transcripts as those which could be readily detected at 1 h p.i. and were not inhibited by CH.
Only two CCV direct repeat region genes were transcribed in the IE stage. In addition to the 1·6 kb IE 3 transcript described previously (Silverstein et al., 1998 ), we also identified a 3 kb IE 1 transcript. Computer analysis of ORFs 1 and 3 and their putative peptide sequences using the FastA and TFastA (Pearson & Lipman, 1988
) programs found no sequence homology between the two genes, and no significant homology to any known DNA or protein sequences. ORF 1 encodes a protein with a predicted molecular mass of 93·5 kDa and an isoelectric point (pI) of 9·97. The protein encoded by ORF 3 has a predicted molecular mass of 32·3 kDa and a pI of 4·26, and a predicted helixturnhelix motif, a potential DNA-binding site, near the amino terminus (amino acids 827). The potential DNA-binding site within the ORF 3 protein supports the possibility that this protein may be involved in regulation of viral transcription, similar to other herpesvirus IE proteins.
Most of the genes within the CCV direct repeat region were expressed during the early stage of transcription (Table 1). Among these early genes, ORF 5 encodes the CCV thymidine kinase (Hanson & Thune, 1993
), and ORF 14 encodes a putative protein kinase. Two of the genes encode putative zinc-binding proteins (ORFs 9 and 12) and three encode potential membrane proteins (ORFs 6, 7 and 8). The study indicates that ORF 12 encodes early and late transcripts, since they were not detected by 1·0 h p.i. and are inhibited by CH block (Fig. 4
). This result conflicts with a previous finding indicating that ORF 12 is an IE gene (Huang & Hanson, 1998
). In the latter study, IE RNA was isolated following CH block for 8 h, beyond the time when the leak in the CH block appears to begin.
Only one gene in the direct repeat region, ORF 10, was exclusively transcribed during the late stage. In addition to this late gene, ORFs 4, 7, 11 and 12 encoded both late and early transcripts. ORFs 7 and 10 encode putative membrane proteins and may be structural virion peptides. ORFs 11 and 12 are related to ORF 9 and encode putative zinc-binding proteins. The function of the protein encoded by ORF 4 is unknown.
All ORFs within the CCV direct repeat region have at least one potential polyadenylation site near their 3' ends, or share such a site with a downstream ORF suggesting 3'-coterminal transcription (Fig. 1; Davison, 1992
). This study demonstrates that several CCV direct repeat genes encode multiple RNA species some of which are apparently transcribed as 3'-coterminal families. The results confirm that two sets of genes, ORFs 5 and 6 as well as ORFs 8 and 9, encode bicistronic transcripts, as previously reported (Silverstein, 1995
, 1998
). ORFs 2 and 3 also appear to encode a 2·9 kb bicistronic transcript. Transcription of ORFs 10, 11, 12 and 13 is particularly complex, encoding multiple early and late polycistronic RNAs (Table 1
). Further analysis including mapping of the 5' and 3' ends of individual transcripts will be required to confirm these polycistronic RNA species.
CCV infection of channel catfish provides a useful natural model in which to study the molecular mechanisms involved in herpesvirus pathogenesis and latency. CCV DNA can be detected in tissues of acutely and latently infected catfish (Gray et al., 1998 , 1999
). Studies are in progress to characterize CCV gene expression in acutely infected fish and to identify latency associated transcripts in tissues of latently infected fish. The characterization of CCV transcription provides a foundation for the further investigation of the molecular basis of CCV pathogenesis and latency.
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Acknowledgments |
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References |
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Received 7 February 2000;
accepted 2 May 2000.
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