The authors have declared that no competing interests exist.
Conceived and designed the experiments: DP MG. Performed the experiments: MG PV AC HCB ES MD. Analyzed the data: MG SP DP. Contributed reagents/materials/analysis tools: CL. Wrote the paper: SP DP.
Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5’UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3’UTR. Despite the weak requirement for a long 5’UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation.
Eukaryotic mRNA translation initiation occurs for the vast majority of cellular mRNAs by scanning of the 5' untranslated region (UTR) by the small ribosomal subunit in complex with the initiator methionine-tRNA and additional translation initiation factors [
Scholars believe that eIF4E recognition of the mRNA 5' cap structure is the first step toward mRNA loading on the ribosomal small subunit. The presence of a 5' cap enhances translation of mRNA 3- to 30-fold [
The 5’UTR length is also important [
Certain mRNA molecules are translated by mechanisms that do not conform to this conventional scanning-initiation model but instead use a specific mechanism. Well characterized examples include the so-called internal ribosome entry sites (IRES) [
The 3'UTRs bear specific, known sequences involved in controlling protein expression [
Group A Rotavirus (RVA) mRNA molecules are capped and bear short 5' and 3’UTRs. As exemplified by the RF rotavirus strain, 5’UTRs (average* 30 ±8) vary from 9 (gene 4) to 50 nt (gene 3), and the 3'UTRs (average* 67 ±30) vary from 17 (gene 1) to 182 (gene 10) (*not considering the gene 11 ORF2 that encodes NSP6, which is not expressed by all viruses). The RVA 5’UTRs begin with a conserved GGCU sequence and 3’UTRs end with the conserved GACC sequence. Remarkably, rotavirus mRNAs are not polyadenylated; however, they are well-translated during infection.
The viral non-structural protein NSP3 is bound to the 3’ end of the viral mRNA during infection [
The NSP3-stimulated translation requirement for viral protein production in cell-culture-adapted rotavirus strains that grow on poorly differentiated epithelial cells (MA104) has been questioned [
To decipher the roles of the rotavirus mRNA 5' and 3’UTRs and the NSP3 domains in NSP3-dependent translation, we first constructed an in vivo assay using electroporation of in vitro-synthesized reporter mRNA in cells transiently expressing NSP3. This assay was then used to define the minimal 5’ and 3’UTRs required for efficient translation of rotavirus-like mRNA and to address the dependence of RVA mRNA translation on the "Kozak" environment.
Our assay uses three types of plasmid vectors (
The NSP3 expression vector was transfected as DNA in BSRT7 cells. The capped reporter mRNA ending with the canonical GACC (R-RNA) or non-canonical (Nc-RNA) terminal sequence was electroporated with the standard RNA (S-RNA).
To quantify the relative contributions of either the 5’ cap or NSP3 on rotavirus-like mRNA translation, capped and uncapped R-RNA were synthesized and used in our assay in the presence or absence of NSP3.
BSRT7 cells expressing NSP3 or eGFP(-) were electroporated with capped (left) or uncapped (right) reporter rotavirus-like mRNA molecules and the standard mRNA.
The 3' end differs from the 3’ GACC canonical sequence in a few rotavirus genes. In the SA11-4F strain, the 3’ end of gene 7 (encoding NSP3) is GGCC and that of gene 5 (encoding NSP1) is GAACC whereas gene 5 from the RRV strain ends with GGCC [
It has been suggested that NSP3-dependent translation of viral mRNA is solely due to viral mRNA 3' end protection by NSP3 binding and that an interaction between NSP3 and eIF4G is not required [
If NSP3 is required to translate rotavirus mRNA, how can NSP3-encoding mRNA be translated at the onset of infection when NSP3 is not present? To answer this frequently asked question, we constructed a self-stimulating reporter mRNA (
The above-described results show that our assay is functional and that it recapitulates known properties of NSP3-dependent translation, which requires a cap, a 3' GACC sequence [
We questioned whether rotavirus gene four 5’UTR-mediated translation operates in accordance with the standard model of translation initiation based on an examination of the rotavirus gene 4 (encoding the spike protein VP4) 5’UTR. This UTR is only 9 nt-long (from the first G to the initiation codon AUG), whereas an initiating ribosome with the AUG codon in the P site protects 12 to 17 nt on the 5’ side [
To test whether the 9 nt of gene 4 5'UTR facilitate conventional translation initiation, the full-length 5’UTR from the gene 4 RF was placed upstream of the Rluc ORF to produce the cap-RF04-RLuc-RF06-GACC reporter mRNA (
BSRT7 cells expressing NSP3 (NSP3; black bars) or no NSP3 (NSP3-KO) (wo. NSP3; white bars) were electroporated with the indicated capped R-RNA molecule (lines 1–6) or capped -p(A)-RNA molecule (lines 7–10) and the standard RNA. The
The initiation codon context largely influences fidelity and efficiency in translation initiation. A weak AUG context promotes ribosomal leaky scanning and downstream initiation.
The first initiation codon in gene 4 is in a good Kozak context (
BSRT7 cells expressing NSP3 (black bars) or no NSP3 (NSP3-KO) (wo. NSP3 white bar) were electroporated with the standard RNA and with capped R-RNA (lines 1–6) with 9- (lines 1–3,6) or 5- (lines 4,5) nt-long 5’UTRs with different AUG (bold and italicized) contexts. The -3 and +4 positions are boxed, and the nucleotides that differ from the wild type (line1) are underlined. The
Interestingly, translation of the reporter mRNA with the gene four 5’UTR was more sensitive to the presence of NSP3 than the previously used reporter mRNA because translation decreased 25-fold without NSP3; however, reporters bearing the gene11 5'UTR typically yield a 5-fold decrease (
The above results (
Reporter mRNA with a 5’UTR that is sufficiently long (gene 11; 21 nt) to accommodate conventional scanning-dependent translation initiation or with the short gene 4 5'UTR was combined with the gene 6 or 4 3'UTR and used in our translation assay (
BSRT7 cells expressing NSP3 were electroporated with the standard RNA and capped R-RNA with the 5'UTR from gene 11 (lines 1,2) or 4 (lines3,4) combined with the 3’UTR from gene 6 (lines 1,3) or 4 (lines 2,4). The
Reducing the 5'UTR to 5-nt did not decrease translation combined with the wild-type 35-nt 3'UTR of gene four (lines 1 and 7). However, translation was slightly weaker when the 5-nt 5'UTR was associated with a shuffled 35-nt 3'UTR (lines 7 and 8).
Thus, whether the reporter mRNA was efficiently translated with the short (9 nt) 5’UTR of gene four was not due to the RNA sequences in the 5' or 3’UTRs.
We constructed an in vivo assay to study the role of the rotavirus NSP3 and rotavirus mRNA non-coding sequences on rotavirus-like mRNA translation. We improved our [
Using this assay, we show that NSP3 expression enhances rotavirus-like mRNA translation. Capped and uncapped mRNAs translation being stimulated by NSP3 to the same extend (
However, synergy between the cap structure and NSP3 was observed, and the presence of a cap or of NSP3 stimulates translation 5-fold, but the presence of both NSP3 and a cap stimulates translation more than 40-fold. This synergy is comparable to the synergy observed between the cap and PABP on polyadenylated cellular mRNA [
R-RNA translation is enhanced even with a small quantity of NSP3 barely detectable by a western blot (
Interestingly, slightly but significantly enhanced Nc-RNA GGCC translation was observed in cells expressing NSP3-SA versus cells lacking NSP3 (
Using the "T2A" reporters, we show positive feedback between NSP3 and its mRNA. During rotavirus infection, such feedback would facilitate rapid accumulation of the first NSP3 molecules, and once NSP3-dependent translation is triggered, NSP3 is available for other viral mRNA molecules. In the SA11 strain, which does not benefit from the positive feedback for gene 7 mRNA translation (due to gene 7 non-canonical GGCC 3' end), the sustained pace of viral mRNA synthesis might facilitate generation, albeit belatedly, of a sufficient quantity of NSP3.
Interestingly, NSP3-mediated enhanced translation varies depending on the 5' rotavirus UTR (5-fold with the R-RNA with the gene 11 5'UTR used in Figs
Using mutants of the NSP3 RNA- or eIF4G- binding domain, we show that both domains do not function properly when separated, but they must lie in the same polypeptide to efficiently stimulate translation, which eliminates the hypothesis that NSP3 includes two independent functions [
Our assay facilitated comparison of NSP3- and poly(A)-dependent translation with an emphasis on translation driven by rotavirus gene 4 UTRs. Rotavirus gene 4 is remarkable because it is efficiently translated despite a short 5'UTR. We show that NSP3-dependent translation is less sensitive to 5’UTR length than poly(A)-dependent translation, but the good environment requirement for the translation initiation codon remains. Translation can initiate at a short 5’UTR (<8-nt) without eIF1 [
Our results using shortened and shuffled 3’UTRs show that the viral 3’UTR can be modified to improve translation; changes in the 3’UTR sequence likely modify the mRNA secondary structure and, consequently, the 3' end accessibility to NSP3. However, a 11-nt 3’UTR strongly decreased reporter translation with the 5’UTR from genes 11 or 4. In this instance, it is highly likely that the distance between the termination codon and 3' end of the RNA bound to NSP3 is not sufficiently large to accommodate a ribosome with a stop codon in the A site; NSP3 would act as a roadblock and render the stop codon inaccessible to the ribosome.
Using our novel in vivo assay, we validated and, more importantly, quantified the roles of NSP3 and regulatory RNA sequences in rotavirus mRNA translation. Our study will be also useful for investigating how other rotavirus proteins or infection-induced physiological changes (e.g., stress and eIF2a phosphorylation) impact rotavirus gene expression.
Baby hamster kidney BSRT7 cells [
The deoxy-oligonucleotides used for the plasmid constructs are listed in
OLIGO NAME | polarity | SEQUENCE | USE |
---|---|---|---|
F7 | RT-PCR for cloning RF07 PCR product digested by NotI and AgeI in NotI-XmaI of priboz | ||
R7 | |||
BsaupRF07 | add BsaI site at the end of the 3'UTR of RF gene 07 | ||
BsaloRF07 | |||
NcoM4RF07up | add NcoI site on the fourth codon of NSP3 ORF | ||
NcoM4RF07lo | |||
F11 | PCR for cloning renilla luciferase ORF between rotavirus UTRs in priboz | ||
R6 | |||
BSAup | add BSA1 site 3' to RF06 UTR (site directed mutagenesis) | ||
BSAlo | |||
NSP3stopup | introduce a stop codon at amino acid postion 7 of NSP3ORF (site directed mutagenesis) | ||
NSP3stoplo | |||
NSP3 mutRNAup | change positions 83-84(RN) of the RNA-binding domain of NSP3 into alanines (site directed mutagenesis) | ||
NSP3mutRNAlo | |||
NSP3mutd4Gup | to introduce a stop codon at position 251 of NSP3; deletion of the eIF4G binding domain (site directed mutagenesis) | ||
NSP3mutd4Glo | |||
NSP3stopup | to introduce a stop codon at position 6 of NSP3 | ||
NSP3stoplo | |||
DIR-SARR07 | RT-PCR of SA11 gene 07. PCR product digested by NotI and XmaI and cloned in priboz | ||
REV-SARR07 | |||
NonaGAACC/Bsa up | to mutate the 3' GACC sequence in GAACC (site directed mutagenesis) | ||
NonaGAACC/Bsa lo | |||
NonaGGCC/Bsa up | to mutate the 3' GACC sequence in GGCC (site directed mutagenesis) | ||
NonaGGCC/Bsa lo | |||
oligodTBSAEcoUp | reverse transcription of polyadenylated dsRNA | ||
BSAEcoUAP | amplification of polyadenylated cDNA | ||
BAMRF6UTR3 | amplification of polyadenylated RF06 3'UTR cDNA | ||
GFPncoup | amplification of eGFP ORFby PCR with a NcoI site at both ends | ||
GFPncolo | |||
GFPT2ANSP3UP | insertion of the T2A coding sequence between BsrGI and AccI sites | ||
GFPT2ANSP3LO | |||
5’ UTR 04RF-9up | cloning gene four (9 nucleotide-long) 5'UTR upstream Renilla luciferase | ||
5’ UTR 04RF-9lo | |||
5’ UTR 04 RF-5up | cloning 5 nucleotide-long 5'UTR upstream Renilla luciferase | ||
5’ UTR 04 RF-5lo | |||
5’ UTR RF-2up | cloning 2 nucleotide-long 5'UTR upstream Renilla luciferase | ||
5’ UTR RF-2lo | |||
AUG Stop 04RF-9up | cloning gene four 5'UTR with stop in place of start codon upstream Renilla luciferase | ||
AUG Stop 04RF-9lo | |||
no kozak +4-9up | cloning gene four 5'UTR with a modified "Kozak" (position +4) upstream Renilla luciferase | ||
no kozak+4-9lo | |||
no kozak-3+4-9up | cloning gene four 5'UTR 9 nucleotide long underlined) with a modified "Kozak" (positions +4 and -3, bold) upstream Renilla luciferase | ||
no kozak-3+4-9lo | |||
no kozak -3+4-5up | cloning gene four 5 nucleotide long 5'UTR with a modified "Kozak" (position -3 and +4) upstream Renilla luciferase | ||
no kozak-3+4-5lo | |||
no kozak-3-5up | cloning gene four 5 nucleotide long 5'UTR with a modified "Kozak" (position +4)upstream Renilla luciferase | ||
no kozak-3-5up | |||
3’ UTR 04 RF-35up | cloning gene four 3'UTR downstream Renilla luciferase | ||
3’ UTR 04 RF-35lo | |||
3’ UTR shuffled-35up | cloning gene four shuffled 3'UTR downstream Renilla luciferase | ||
3’ UTR shuffled-35lo | |||
3’ UTR 04 RF-11up | cloning gene four short(11 nucleotides) 3'UTR downstream Renilla luciferase | ||
3’ UTR 04 RF-11lo | |||
3’ UTR 04RF-20up | cloning gene four short (20 nucleotides) 3'UTR downstream Renilla luciferase | ||
3’ UTR 04RF-20lo | |||
3’ UTR shuffled-24up | cloning shuffled (24) 3'UTR downstream Renilla luciferase | ||
3’ UTR shuffled-24lo | |||
FlucACC65up | to introduce an Acc65I site in Fluc ORF of pGL3 | ||
FlucACC65lo |
To construct the NSP3-RF expression vectors (
Mutations were introduced in the NSP3-RF RNA- and eIF4G-binding domains through site-directed mutagenesis. The amino acids 83(R) and 84(N) in contact with the RNA [
Two negative controls were used; pT7-Ires-eGFP expressed the protein eGFP instead of NSP3, and pT7-ires-NSP3-KO encoded only the first 6 amino acids of NSP3-RF. To generate pT7-Ires-eGFP, the eGFP coding sequence was retrieved from the peGFPN1 (Clontech) as an NcoI-XbaI fragment and cloned into pT7-RF06-Fluc-RF06 [
A schematic representation of the plasmids used to synthesize the reporter mRNAs is presented in
Site-directed mutagenesis (Quick-change, Stratagene) using the oligonucleotide pairs NonaGAACC/Bsa and NonaGGCC/Bsa (up and lo,
To produce a reporter mRNA (pA-RNA) that differs from the R-RNA reporter by only a stretch of adenines, genomic dsRNA purified from RF RVA virions was polyadenylated with
The 5’UTR and initiation codon environment in pT7-RF-Rluc-GACC-Bsa and pT7-RF-Rluc-p(A)-Bsa were modified by cloning pairs of complementary oligonucleotides (5'UTRgene04RF-9up to nokozak-3-5,
The pT7-RF-Rluc-GACC-Bsa 3’UTR was modified by cloning two complementary, annealed oligonucleotides between the BamHI and EcoRI sites (oligonucleotides 3'UTR 04RF-35, shuffled-35) or the XbaI and EcoRI sites (oligonucleotides 3'UTR 04RF-11, 04RF-20 and shuffled-24) of pT7-RF-Rluc-GACC-Bsa.
The standard EMCV Fluc RNA was generated through in vitro transcription of the pEMCV-Fluc plasmid linearized through treatment with the restriction enzyme EcoRI. To produce the pEMCV-Fluc plasmid, the pT7-RF06-Fluc-RF06 Fluc ORF [
The PCR fragments and annealed oligonucleotides were fully sequenced after cloning into the target plasmids. For site-directed mutagenesis, the entire functional (from the T7 promoter to the T7 terminator) unit was sequenced. All plasmid constructs were validated through restriction enzyme mapping.
The eGFP ORF was first introduced in frame with the NSP3 ORF into pribozRF07Nco/Bsa (see above) as an NcoI PCR fragment (using GFPncoup and GFPncolo as primers,
The capped RNA molecules were generated through in vitro transcription of the template plasmids linearized by BsaI using the mMessage mMachine T7ultra kit (Ambion). The DNA was removed through an enzymatic treatment (15 min 37°C) with RNAse-free DNase, and the RNA molecules were purified using MegaClear (Ambion) silica spin columns to eliminate unincorporated cap analogues and nucleotides prior to ethanol precipitation. The purified RNA molecules were quantified using a spectrophotometer (Nanodrop), then controlled using denaturing agarose gel electrophoresis and stored in aliquots at minus 80°C. Uncapped mRNA or IRES mRNA molecules were synthesized using the same linearized templates and T7mega script kit (Ambion), which does not include the ARCA cap analog [
Unless stated otherwise, the reporter mRNA was capped. The reporter mRNA is abbreviated as R-RNA (cap-Rluc-GACC mRNA) for rotavirus-like reporter mRNA, Nc-RNA for rotavirus-like reporter mRNA ending with the non-canonical 3' end GAACC or GGCC and pA-RNA (cap-Rluc-p(A)) for poly(A) mRNA.
The DNA was introduced into BSRT7 by lipofection using Lipofectamine 2000 reagent in accordance with the supplier’s instructions (Life Technologies). Twenty-four hours later, RNA was introduced into BSRT7 cells using a Neon electroporation device (Life Technologies). The cells (106) were suspended in R buffer (Life Technologies) with 50 ng of reporter mRNA (and 1 μg of standard RNA). The conditions for optimal electroporation were determined for the BSRT7 cells as two 20 ms pulses of 1400 V. After electroporation, complete culture medium was immediately added, and the cells were incubated at 37°C.
At the indicated times, the cells were lysed in the passive lysis buffer (Promega) for 15 min and then frozen at -20°C. Luminescence was measured using the Dual-Luciferase-Reporter Assay System (Promega) and a luminometer (Sirius, Berthold). The results are from three experiments conducted in triplicate with three different reporter mRNA preparations. The results were analyzed using Student's two-tailed
Proteins separated using SDS-PAGE were transferred to low-fluorescence PVDF membranes and probed with a rabbit polyclonal antibody against NSP3 (4–150)
A: Schematic representation of the NSP3 expression vector, pT7-ires-NSP3. B: Schematic representations of 1- the vector pT7 RF-Rluc-GACC-Bsa used for in vitro synthesis of the rotavirus-like (R-RNA) reporter mRNA and 2- the vector pT7RF-Rluc-p(A)-Bsa used for in vitro synthesis of the polyadenylated RNA reporter (pA-RNA). The DNA sequences (top and bottom strands) at the start and end of the T7 RNA polymerase transcription unit are indicated. The start site (+1) is indicated, the DNA sequence recognized by the BsaI restriction enzyme is underlined, and the slashes indicate the position of the cuts. The rotavirus 3' consensus sequence (GACC) mutated in the Nc-RNAs vectors is indicated in bold. The 5' and 3' end sequences for the RNA produced by the T7 RNA polymerase (that uses the bottom DNA strand as a template) with the plasmids cut by BsaI are indicated. C: Schematic representation of the vector pT7-ires-Fluc used for in vitro synthesis of the transfection standard RNA (S-RNA). The positions of the restriction sites used for plasmid constructions (see text) are also indicated. pT7: T7 RNA polymerase promoter; terT7: T7 RNA polymerase terminator; IRES: EMCV Internal Ribosome Entry Site; Rluc:
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