Veterinary Molecular Biology, PO Box 173610, Montana State University, Bozeman, MT 59717-3610, USA
Correspondence
Michele Hardy
mhardy{at}montana.edu
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ABSTRACT |
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The GenBank accession number for the nucleotide sequence of gene 5 of bovine rotavirus strain B642 is AF458087.
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Introduction |
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The molecular mechanisms that regulate transcription and subsequent translation of individual rotavirus genes are not well defined. Johnson & McCrae (1989) measured the levels of viral transcripts and encoded proteins and showed that there was quantitative as well as temporal control of rotavirus gene expression. The 11 viral proteins were not synthesized to equivalent levels in infected cells, nor did the levels of individual proteins correspond with the levels of the cognate mRNAs. These observations suggested both transcriptional and translational control of viral gene expression. Furthermore, these data implied that signals in some, if not all, of the 11 viral mRNAs regulated the levels of specific viral proteins. The mechanisms of regulation were not defined.
Few studies have investigated the role that protein synthesis plays in controlling expression of rotavirus genes throughout the replication cycle. A recent study identified a specific interaction between non-structural protein NSP3 and the cellular translation initiation factor eIF4GI (Piron et al., 1998). NSP3 is a sequence-specific RNA-binding protein that binds to the last four to five conserved nucleotides of the 3' end of viral mRNAs (Poncet et al., 1994
). eIF4GI is a component of the eIF4F initiation complex that recognizes the 5' cap structure typical of eukaryotic mRNAs. The interaction between NSP3 and eIF4GI most likely promotes circularization of viral mRNAs to enhance translation, as previously proposed (Piron et al., 1998
; Vende et al., 2000
). At the RNA level, a four-nucleotide translation enhancer element in the 3'-terminal consensus sequence of rotavirus mRNAs has been reported (Chizhikov & Patton, 2000
). Together, these studies provide initial evidence that trans-acting viral proteins and cis-acting signals in viral mRNA contribute to controlling synthesis of rotavirus proteins in infected cells.
We have investigated potential mechanisms of post-transcriptional regulation of rotavirus gene expression by analysing the translational efficiencies of gene 5 and gene 6 mRNAs. These two genes were chosen as models for analysis of rotavirus gene regulation because: (i) both are expressed early in infection; (ii) VP6 is expressed at higher levels than NSP1; and (iii) the mRNAs and ORFs are similar in size, thus minimizing potential variations in expression due to mRNA length. Gene 5 encodes non-structural protein NSP1. The function of NSP1 in infected cells is not clear, but NSP1 appears to be non-essential for replication, because viruses with gene 5 rearrangements that do not synthesize NSP1 have been isolated (Allen & Desselberger, 1985; Biryahwaho et al., 1987
; Kojima et al., 2000
; Pedley et al., 1984
; Taniguchi et al., 1996
). Gene 6 encodes the major inner capsid protein, VP6, which is the subgroup-specific antigen and is required for viral transcriptase activity (Estes & Cohen, 1989
). We examined mRNA accumulation, protein stability and polyribosome distribution of genes 5 and 6 mRNA in rotavirus-infected cells. Our data suggest that the difference in the amounts of NSP1 and VP6 is accounted for by a difference in the translational efficiencies of the mRNA.
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Methods |
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Cloning and sequencing of B641 gene 5.
Gene 5 was cloned by RT-PCR. Transcriptionally active double-layered virus particles were purified by CsCl gradient centrifugation, and viral mRNA was synthesized in vitro as previously described (Hardy et al., 1991, 1992
). Purified mRNA was reverse-transcribed for 3 h with avian myeloblastosis virus reverse transcriptase (Promega) primed with the oligonucleotide 5'-CACGGATCCGGTCACATTTTGCAGGGAGTCTTG-3' complementary to the 3' end of the gene 5 mRNA. Following reverse transcription, mRNA was degraded by alkaline hydrolysis and cDNA was purified by ethanol precipitation in the presence of 0·3 M sodium acetate, pH 5·2. Gene 5 then was amplified with Pfu polymerase (Stratagene) by 30 cycles of PCR with the above primer and the positive-sense primer 5'-CACGGATCCGGCTTTTTTTTATGAAAAGTCTTG-3'. Both oligonucleotides contained BamHI restriction sites for cloning (underlined) and were designed based on consensus sequence alignments of gene 5 sequences of other rotavirus strains. Cycling conditions were as follows: 94 °C for 1 min, 50 °C for 45 s and 72 °C for 2 min. The resultant 1·5 kb PCR product was cloned into pBluescript KS+ (Stratagene). The nucleotide sequence was determined on an ABI 310 Genetic Analyser with BigDye Terminator chemistry. Comparison of the nucleotide sequence with those of other rotavirus strains indicated that gene 5 of B641 is 98 % identical at the nucleotide level to gene 5 of bovine strain RF (Bremont et al., 1987
).
Metabolic labelling of rotavirus proteins.
Viral proteins synthesized in infected cells were labelled as previously described, with modifications (Ericson et al., 1982). MA104 cells were infected at an m.o.i. of 10 in medium lacking methionine and cysteine and containing 5 % FBS. At 4 h post-infection (p.i.), the medium was replaced with medium without FBS and containing 10 µg actinomycin D ml-1 and 40 µCi TRAN35S-label ml-1 (ICN). Cultures were harvested at the indicated times by gentle rocking in RIPA buffer (150 mM NaCl, 1 % sodium deoxycholate, 1 % Triton X-100, 0·1 % SDS, 10 mM Tris/HCl, pH 7·2, 1 % Trasylol). Labelled proteins were resolved by SDS-PAGE and visualized by autoradiography.
Pulse-chase labelling was performed as previously described (Ericson et al., 1982) and as outlined above, except that following a 30 min pulse with 40 µCi TRAN35S-label ml-1, cultures were chased with medium containing 400xunlabelled methionine and cysteine and 100 µg cycloheximide ml-1. Cultures were chased for the indicated times, harvested in RIPA buffer and radiolabelled proteins were analysed by SDS-PAGE. The identity of the band in MA104 cells infected with rotavirus under these conditions has been confirmed by reactivity with an anti-NSP1 monoclonal antibody. Radioactive protein bands were quantified on a Bio-Rad Molecular Imager F-X with Quantity One software.
RNA extraction and Northern blot hybridization.
Total RNA was harvested from mock-infected or B641-infected cells by Trizol (InvitrogenLife Technologies) extraction following the procedures recommended by the manufacturer. RNA was electrophoresed in 1·2 % agarose gels containing 6 % formaldehyde and transferred to nylon membrane by capillary blotting. Radiolabelled probes were prepared by nick-translation of specific gene fragments, which were digested and purified from plasmid vectors. The plasmid containing SA11 gene 6 in the pSP65 vector (Promega) was a gift from M. K. Estes (Baylor College of Medicine, Houston, Texas, USA) (Estes et al., 1984). This plasmid was transformed into E. coli DH5
cells and mid-scale plasmid purifications were performed by standard methods (Sambrook & Russell, 2001
). The specific activity of the probes was determined by liquid scintillation counting and care was taken to ensure that the specific activities were comparable between probes for different genes and different experiments to allow quantitative interpretation of the data. Hybridizations were performed at 42 °C in the presence of 50 % formamide. Radioactive signals on the blots were quantified on a Bio-Rad Molecular Imager F-X with Quantity One software.
Polyribosome analysis.
All polyribosome analyses were performed several times and representative data are shown. Cytoplasmic extract preparation and polyribosome analysis were performed and interpreted as previously described (Detjen et al., 1982; Kaspar et al., 1992
; Lodish, 1971
; Walden & Thach, 1986
; White et al., 1990
). MA104 cells were mock-infected or infected with B641 at an m.o.i. of 510. At the indicated times p.i., the cells were treated with 100 µg cycloheximide ml-1 to arrest ribosome transit. Cells were collected from the dishes with a 1x times; trypsin solution containing 100 µg cycloheximide ml-1. Cells were swollen in low-salt buffer (20 mM Tris/HCl, pH 7·4, 10 mM NaCl, 3 mM MgCl2) and lysed with low-salt buffer containing 1·2 % Triton N-101 and 200 mM sucrose and nine strokes of a Dounce homogenizer. Cell nuclei were removed by brief centrifugation and cell lysates were layered onto 0·51·5 M sucrose gradients prepared in low-salt buffer. The gradients were centrifuged for 58 min at 159 000 g in a Beckman SW55 rotor and fractionated with an ISCO density gradient fractionator with an absorbance monitor at 254 nm. RNA was extracted from 500 µl fractions with 1 : 1 phenol/chloroform, then with chloroform and finally precipitated with 250 mM NaCl, 20 µg glycogen ml-1 and ethanol. The RNA from each fraction was electrophoresed through 1·2 % agarose/6 % formaldehyde gels and subjected to Northern blot hybridization analysis as described above.
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Results |
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Discussion |
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NSP1 is the least conserved protein in the rotavirus genome and its function in the replication cycle is not known. The cysteine-rich N-terminal domain of NSP1 contains a putative zinc finger motif similar to those in eukaryotic transcription factor TFIIIA and this region is required to bind the 11 viral mRNAs in vitro (Hua & Patton, 1994; Hua et al., 1993
). The low level of expression of NSP1 persists throughout the replication cycle (Ericson et al., 1982
). The number of functional molecules of NSP1 in infected cells is most likely tightly controlled, consistent with its role as a regulatory protein. Interestingly, NSP1 appears to be dispensable for virus replication, as mutant rotavirus strains with rearrangements in gene segment 5 that do not encode NSP1 are viable in cell culture (Biryahwaho et al., 1987
; Hundley et al., 1985
, 1987
; Pedley et al., 1984
; Taniguchi et al., 1996
). Isolation of such mutants provides further support for the role of NSP1 as a regulatory protein, in that viral strains defective for NSP1 expression display a small-plaque phenotype compared with their wild-type counterparts.
In contrast to a regulatory function for NSP1, VP6 forms the major inner capsid layer of the mature virus particle. The mature virion contains 260 trimers of VP6 (Prasad & Chiu, 1994). If a single infected cell releases 5060 p.f.u. (Johnson & McCrae, 1989
), then the number of molecules of VP6 that must be synthesized falls between 39 000 and 46 800. In fact, this number is probably much higher, given that this calculation assumes a particle to p.f.u. ratio of 1 and the particle to p.f.u. ratio for rotavirus has been reported to be as high as 100 : 1. In either case, VP6 must be synthesized in high amounts and the mRNA encoding VP6 must be translated more efficiently than that encoding NSP1.
Cis-acting signals in rotavirus mRNAs and trans-acting viral proteins that function in regulating rotavirus gene expression have only recently come under study. Rotavirus belongs to the family Reoviridae, and studies of translation of reovirus mRNA have provided a significant amount of original data on the selectivity of the cellular translational apparatus and the features of a viral mRNA that might contribute to such selectivity (Detjen et al., 1982; Golini et al., 1976
; Lawson et al., 1988
; Ray et al., 1985
; Walden et al., 1981
). Walden et al. (1981)
reported hierarchical translation efficiency of mRNAs transcribed from reovirus gene segments. A series of studies led this group to propose that translation rates in reovirus-infected cells are regulated by competition of host and viral mRNAs for limiting translation factors, and that the competitive ability of a mRNA was determined by its ability to efficiently recruit translation initiation factors (Brendler et al., 1981a
, b
; Godefroy-Colburn & Thach, 1981
; Walden et al., 1981
). Direct competition between rotavirus gene 5 and gene 6 mRNA was not addressed in this study.
Structural features in the 5'UTR of a mRNA that can dictate its translation efficiency are well established and include cap-proximal secondary structure and length of the UTR (Kozak, 1984, 1987
, 1988
). The 5'UTR of gene 5 of B641 is 31 bases, longer than the 23 nucleotide 5'UTR of gene 6. Additional ribonucleotides in the gene 5 5'UTR might contribute to a secondary structure that is inhibitory to translation initiation. However, the 5'UTRs of the 11 rotavirus gene segments range from 9 to 49 nucleotides and the lengths of the UTRs do not correlate with the level of the encoded protein. For example, VP3 is a structural core protein synthesized at low but detectable levels in infected cells and has a 49-base 5'UTR. Though these observations do not dismiss a role for the 5'UTR in translational efficiencies of rotavirus mRNA, there must be additional components of the regulatory mechanism. Such mechanisms include a potential interactive synergy with the 3'UTR and differing affinities of RNA-binding proteins, viral or cellular, for 5' and 3' ends of each viral mRNA. We also noted a short non-overlapping upstream ORF immediately preceding the start codon in gene 5 of B641 and of bovine strains RF (Bremont et al., 1987
), UK (Hua & Patton, 1994
) and A44 (Kojima et al., 1996
) that could negatively affect the efficiency of translation of this mRNA. Translation control by upstream ORFs has been documented for a number of viral and cellular mRNAs, and in some cases reduces expression of the downstream ORF (reviewed in Geballe & Sachs, 2000
).
Evidence continues to accumulate that implicates the 3'UTR in translational control of gene expression in both cellular and viral mRNAs (Gallie, 1996; Gallie & Kobayashi, 1994
; Hann et al., 1997
; Leathers et al., 1993
; Tanguay & Gallie, 1996
; Zeyenko et al., 1994
). Tanguay & Gallie (1996)
reported that a longer 3'UTR on a mRNA lacking a poly(A) tail increased the translational efficiency of a luciferase reporter mRNA and this effect was sequence-independent and gene-independent. Rotavirus mRNAs do not have poly(A) tails and unlike the comparison between the 5'UTR of genes 5 and 6, the 3'UTRs are quite different. The 3'UTR of gene 5 is 50 bases long compared with 139 bases for gene 6. The length of the 3'UTR may play a more important role in regulating translation efficiency of rotavirus mRNA than the 5'UTR. A mechanism for how the length of a 3'UTR enhances the translation efficiency of an mRNA has been proposed (Tanguay & Gallie, 1996
). It was suggested that following termination and dissociation of the 80S ribosome, the 40S ribosomal subunit continues to transit the 3'UTR. Therefore, mRNAs with a longer 3'UTR would have a higher local concentration of ribosomal subunits available to reinitiate translation of the same mRNA. This seems a plausible explanation for the difference in translational efficiencies of genes 5 and 6, certainly under conditions of competition for limiting translation factors early in the rotavirus replication cycle.
A recent report identified a conserved four-nucleotide enhancer sequence (GACC) present at the 3' termini of rotavirus genes that enhanced expression of a luciferase reporter gene when RNA encoding this sequence was transfected into rotavirus-infected cells (Chizhikov & Patton, 2000). These four nucleotides, when present at the 3' terminus of rotavirus mRNA, bind NSP3 (Poncet et al., 1993
), and it was proposed that variation in this sequence could negatively affect viral protein expression by altering binding of NSP3 (Patton et al., 2001
). The data reported thus far were gained from analysis of luciferase reporter genes and whether such variation in the 3'-terminal sequence causes a decrease in synthesis of the native protein in the context of an infection remains to be determined. Regardless, these data suggest an intriguing role for the 3'UTR in regulation of protein synthesis programmed from viral mRNA in rotavirus-infected cells. Future studies will address the role of sequences and structures of rotavirus UTRs in regulating viral protein synthesis and trans-acting factors that function in regulating expression levels of rotavirus genes throughout the replication cycle.
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ACKNOWLEDGEMENTS |
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Received 2 May 2002;
accepted 23 August 2002.
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