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.2015 Jun;89(11):5919-34.
doi: 10.1128/JVI.00463-15. Epub 2015 Mar 25.

The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection

Affiliations

The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection

Craig H Kerr et al. J Virol.2015 Jun.

Abstract

Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells.

Importance: Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA into Drosophila cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5' untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1
FIG 1
RT-PCR and sequence of the CrPV 5′ UTRs from different viral stocks. (A) RT-PCR analysis of the CrPV 5′ UTRs from cells that were infected with different CrPV stocks from circa 2006, 2009, or 2013 for 24 h. (B) Sequence of the first 500 nucleotides of the 5′ UTR from CrPV-2013, which contains the tandem 196-nt duplication (the first sequence in italics and the repeated sequence in boldface).
FIG 2
FIG 2
Translational control mediated by the duplication in the CrPV-2 5′ UTR. (A) Schematics of reporter constructs. Minigenome reporter RNAs (top) contain Renilla and firefly luciferase genes in lieu of the CrPV ORF1 and ORF2, respectively, but maintain the untranslated regions of the CrPV genome. The bicistronic reporter RNA (bottom) contains a 5′ m7G cap structure and a 3′ poly(A) tail. Renilla luciferase is translated via 5′-end scanning-dependent translation while firefly luciferase is IRES dependent. Gray triangles represent the duplicated sequence element. (B)In vitro translation.In vitro-transcribed minigenome RNA containing the 5′ UTR with (5′ UTR+Dup) or without (5′ UTRΔDup) the 196-bp duplication was incubated in mock- or CrPV-infected S2 cell extracts for 30 min at 30°C. The Renilla and firefly luciferase activities monitor 5′ UTR- and IGR IRES-dependent translation, respectively. Luciferase activity is normalized to that of the minigenome reporter construct containing the 5′ UTRΔDup. (C) Translational activity in S2 cells. S2 cells, transfected with the indicated minigenome RNAs for 2 h at 25°C, were either mock or CrPV infected (MOI of 10) for 6 h. Luciferase activity is normalized to that of the minigenome reporter construct containing the 5′UTRΔDup. (D) IRES activity. Bicistronic RNAs containing either the CrPV 5′ UTR+Dup or 5′ UTRΔDup or an empty intergenic region were incubated in mock- or CrPV-infected S2 cell extracts for 30 min at 30°C. Renilla luciferase measures scanning-dependent translation while firefly luciferase measures IRES-mediated translation. The ratios of firefly to Renilla luciferase activities were normalized to the IGR IRES activity. Shown are averages from at least three independent experiments (± standard deviations).
FIG 3
FIG 3
Construction and map of the full-length cDNA clones CrPV-2 and CrPV-3. (A) RNA from CsCl-purified CrPV was reverse transcribed using an oligo(dT) primer. Primers P1 and P3 were used to clone the CrPV genome without the 5′ UTR into pAC5.1/V5 His B while primers P2 and P3 containing KpnI and SacI restriction sites, respectively, were used to amplify the CrPV genome. CrPV contains an endogenous HindIII site in ORF1. Therefore, to clone the 5′ UTR, both the P1/P2 amplicon and pCrPV-Δ5′ UTR were digested with HindII and KpnI. The resulting fragments were ligated together to generate pCrPV-1. A T7 polymerase promoter was added to pCrPV-1 via primer incomplete polymerase extension (PIPE) to create pCrPV-2. To create pCrPV-3, a PCR fragment derived from the 2006 stock of CrPV was inserted into the KpnI and HindIII sites of pCrPV-2 followed by a T7 promoter. (B) At top the organization of the CrPV-2 and CrPV-3 cDNA genomic sequences showing all annotated proteins is shown. Arrowheads indicate either synonymous (black) or nonsynonymous (white) changes that differ from the published CrPV sequence (GenBank accession number NC_003924). Insertion-deletion events are indicated by an asterisk. A gray triangle indicates the location of the 196-nt duplication in CrPV-2. See Table 1 for specific nucleotide changes. The sequences of the 5′ and 3′ ends of the cloned viral genomes are shown at the bottom of the panel. Nucleotides corresponding to the viral genome are bolded. Arrows indicate the beginning and end of thein vitro transcript obtained from using T7 polymerase with Ecl136II-linearized plasmid.
FIG 4
FIG 4
CrPV-2 and CrPV-3 RNAs produce viral proteinsin vitro. In vitro-synthesized RNA from CrPV-2, CrPV-3, CrPV-2-ORF1-STOP, or CrPV-2-ORF2-STOP was incubated in Sf-21 translation extracts for 2 h at 30°C in the presence of [35S]methionine-cysteine. Reaction products were resolved by SDS-PAGE, and radioactive proteins were visualized by phosphorimager analysis. As a control, lysates from [35S]methionine-cysteine-labeled mock- and CrPV-infected S2 cells were resolved by SDS-PAGE in parallel (lanes 1 and 2). Shown are representative gels from at least three independent experiments.
FIG 5
FIG 5
Transfection ofin vitro-transcribed CrPV-2 and CrPV-3 RNAs in S2 cells. (A) Phase-contrast images of S2 cells mock transfected, CrPV infected (MOI of 0.4), or transfected within vitro-transcribed CrPV-2, CrPV-3, CrPV-2-ORF1-STOP, or CrPV-2-ORF2-STOP RNA at 48 h posttransfection (hpt). (B) Northern blots of CrPV RNA genome from RNA isolated from CrPV-infected S2 cells or cells transfected with the indicated CrPV genomic RNA. Methylene blue staining of blots are shown below. (C) RT-PCR analysis of CrPV negative-strand (−) synthesis from RNA of cells transfected with the indicated CrPV genomic RNA or CrPV-infected at 24 hpt. Results of reactions in the presence (+RT) or absence (−RT) of reverse transcriptase are shown. (D) Western blots of viral 3CD (ORF1) and VP2 (ORF2) from lysates of cells transfected with the indicated CrPV genomic RNA or CrPV infected at 48 hpt.
FIG 6
FIG 6
Transfected CrPV RNA clones produce infectious viral particles. (A) Negatively stained electron micrographs of viral particles purified from CrPV-infected S2 cells or from S2 cells transfected with CrPV-2 or CrPV-3 RNA at 48 h posttransfection. Scale bar, 100 nm. (B) Transwell assay. S2 cells transfected with the indicatedin vitro-transcribed CrPV genomic RNA or infected with CrPV at an MOI of 0.4 for 24 h were seeded on a 0.4-μm transwell insert, which overlays naive S2 cells at the bottom of the well. Cells were then incubated for 24 or 48 h. Cells were analyzed by indirect immunofluorescence using anti-CrPV VP2, and the nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). Shown are representative images from at least three independent experiments.
FIG 7
FIG 7
S2 Cells transfected within vitro-transcribed CrPV-2 and CrPV-3 RNAs accumulate infectious virions over time. A total of 2.5 × 106 S2 cells were transfected with 3 μg of the indicatedin vitro-transcribed genomic RNA or infected with CrPV (MOI of 0.4). Titers were measured as described in Materials and Methods at 3, 12, 24, and 48 hpt. Shown are averages from at least three independent experiments (± standard deviations). *,P < 0.05; **,P < 0.005; ND, not detected.
FIG 8
FIG 8
CrPV infectious clones can infect Drosophila S2 cells devoid of Drosophila C virus, flock house virus, and Drosophila X virus. (A) Detection of Drosophila C virus (DCV), flock house virus (FHV), and Drosophila X virus (DXV) by RT-PCR analysis in UBC S2 cells, Kc167 cells, Invitrogen S2 cells, and S2 cells from the Drosophila Genomics Resource Center. (B) Northern blots of CrPV RNA genome from RNA isolated from Kc167 cells and S2 cells from Invitrogen that were transfected with the indicated CrPV genomic RNA. The presence (+) or absence (−) of DCV, FHV, and DXV is indicated for each cell line. Methylene blue staining of blots is shown below.
FIG 9
FIG 9
Injection of CrPV-2 and CrPV-3 virions into adult Drosophila melanogaster flies. VirginIso w1118 flies (10 males and 10 females) were injected intrathoracically with 5,000 FFU of CrPV, CrPV-2, CrPV-3, or UV-inactivated CrPV-2 and CrPV-3 or with PBS. Subsequently, flies were flipped onto standard medium, and survival was monitored daily.
FIG 10
FIG 10
The CrPV-2 5′ UTR reduces viral fitness. (A) S2 cells were mock infected or infected with CrPV-2 or CrPV-3 (MOI of 10). Cells were metabolically labeled with [35S]methionine-cysteine for the last 30 min of infection. Lysates were subjected to SDS-PAGE, immunoblotting, or Northern blot analysis. Accumulation of viral RNA was monitored by probing for CrPV RNA genome. Expression of CrPV ORF1 was assessed by anti-RdRp antibody. (B) S2 cells were infected with CrPV-2 or CrPV-3 (MOI of 1), and viral titers were measured as described in Materials and Methods at 6, 9, 12, and 24 h p.i. **,P < 0.005; ***,P < 0.0005. Shown are averages from at least three independent experiments (± standard deviations). (C) RT-PCR analysis of the CrPV-2 and CrPV-3 5′ UTRs after serial passage in S2 cells. Cells were transfected with either CrPV-2 or CrPV-3 RNA and virus was harvested (passage 0 [P0]). Cells were then infected at an MOI of 10 and passaged in S2 cells 5 times (P1 to P5). At each passage, RNA was isolated and subjected to RT-PCR analysis.
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