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Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

Rainey SM, Martinez J, McFarlane M, Juneja P, Sarkies P, Lulla A, Schnettler E, Varjak M, Merits A, Miska EA, Jiggins FM, Kohl A - PLoS Pathog. (2016)

Bottom Line: Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA.Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity.Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway.

View Article: PubMed Central - PubMed

Affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

ABSTRACT
The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.

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The effect of Wolbachia on SFV replicon and transreplicase activity.(A, B) Jw18Wol and Jw18Free cells were transfected with in vitro transcribed SFV1(3F)RLuc-SG-FFLuc RNA, and both RLuc (A) and FFLuc (B) activity was measured 24 h post transfection (hpt). Graphs indicate mean fold change of measurements of luciferase activity where activity in Jw18Wol cells is taken as 1 and represent three independent experiments carried out in triplicate. RLuc activity represents translation of genome RNAs whereas FFLuc indicates translation of the subgenomic mRNA. Error bars represent standard error of mean. Stars indicate significance where P = <0.05 in T-Test analysis. (C, D) Jw18Wol and Jw18Free cells were transfected with two plasmids: one expressing SFV replicase proteins (wt or mutant, under the control of the D. melanogaster Actin promoter) and one expressing viral template and both FFluc (C) and Gluc (D) activity were measured 24 hpt. FFLuc activity represents translation of RNA produced from the Actin promoter (and to some extent the genomic promoter) and Gluc activity represents translation of RNA produced from the subgenomic promoter following replication. Graphs represent relative luciferase activity and represent three independent experiments carried out in duplicate. Stars indicate significance where P = <0.01 in T-Test analysis.
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ppat.1005536.g003: The effect of Wolbachia on SFV replicon and transreplicase activity.(A, B) Jw18Wol and Jw18Free cells were transfected with in vitro transcribed SFV1(3F)RLuc-SG-FFLuc RNA, and both RLuc (A) and FFLuc (B) activity was measured 24 h post transfection (hpt). Graphs indicate mean fold change of measurements of luciferase activity where activity in Jw18Wol cells is taken as 1 and represent three independent experiments carried out in triplicate. RLuc activity represents translation of genome RNAs whereas FFLuc indicates translation of the subgenomic mRNA. Error bars represent standard error of mean. Stars indicate significance where P = <0.05 in T-Test analysis. (C, D) Jw18Wol and Jw18Free cells were transfected with two plasmids: one expressing SFV replicase proteins (wt or mutant, under the control of the D. melanogaster Actin promoter) and one expressing viral template and both FFluc (C) and Gluc (D) activity were measured 24 hpt. FFLuc activity represents translation of RNA produced from the Actin promoter (and to some extent the genomic promoter) and Gluc activity represents translation of RNA produced from the subgenomic promoter following replication. Graphs represent relative luciferase activity and represent three independent experiments carried out in duplicate. Stars indicate significance where P = <0.01 in T-Test analysis.

Mentions: As Wolbachia inhibits viral infection or subsequent processes as early as 7 hpi it could be hypothesised that entry of the virus into cells is inhibited, leading to a significant reduction in the number of subsequent replication complexes. In order to test this hypothesis we bypassed viral entry and analysed early translation and replication by transfection of in vitro transcribed capped SFV1(3F)RLuc-SG-FFLuc replicon RNA (Fig 1B). In this SFV-derived replicon RNA, an open reading frame (ORF1) encoding RLuc is fused to the non-structural nsP3 and the second, structural ORF(2) has been deleted and replaced with the FFLuc ORF (see Materials and Methods). Alphavirus gene expression occurs in separate phases which are linked to replicative processes. Initially ORF1 is translated from the RNA genome giving rise to the nsP proteins, which carry out replicative functions. Then a switch from translation to replication occurs leading to production of a full length antisense copy of the genome, the antigenome. Antisense RNA (which most likely exists in a duplex with the original positive-strand genome) is used as a template for synthesis of new genomes; in addition it carries an internal promoter sequence that directs transcription of a subgenomic mRNA encoding the structural proteins (Fig 1) [39]. Thus, expression of structural proteins (or FFLuc marker, Fig 1B) takes place only from these subgenomic RNAs i.e. is fully dependent on the replication process. In contrast, RLuc can be produced both by directly translating the replicon that was transfected into the cells as well as by translating new full-length positive strands, generated during RNA replication. Furthermore, in the absence of structural proteins no new virus particles can be formed preventing the spread of infection. It was found that Wolbachia results in a significant inhibition of early translation and/or replication independent of normal viral entry, with both RLuc and FFLuc readouts being significantly lower in Jw18Wol cells compared to Jw18Free cells (Fig 3A and 3B). We cannot rule out that Wolbachia may also have an effect on entry which we do not observe in these assays.


Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

Rainey SM, Martinez J, McFarlane M, Juneja P, Sarkies P, Lulla A, Schnettler E, Varjak M, Merits A, Miska EA, Jiggins FM, Kohl A - PLoS Pathog. (2016)

The effect of Wolbachia on SFV replicon and transreplicase activity.(A, B) Jw18Wol and Jw18Free cells were transfected with in vitro transcribed SFV1(3F)RLuc-SG-FFLuc RNA, and both RLuc (A) and FFLuc (B) activity was measured 24 h post transfection (hpt). Graphs indicate mean fold change of measurements of luciferase activity where activity in Jw18Wol cells is taken as 1 and represent three independent experiments carried out in triplicate. RLuc activity represents translation of genome RNAs whereas FFLuc indicates translation of the subgenomic mRNA. Error bars represent standard error of mean. Stars indicate significance where P = <0.05 in T-Test analysis. (C, D) Jw18Wol and Jw18Free cells were transfected with two plasmids: one expressing SFV replicase proteins (wt or mutant, under the control of the D. melanogaster Actin promoter) and one expressing viral template and both FFluc (C) and Gluc (D) activity were measured 24 hpt. FFLuc activity represents translation of RNA produced from the Actin promoter (and to some extent the genomic promoter) and Gluc activity represents translation of RNA produced from the subgenomic promoter following replication. Graphs represent relative luciferase activity and represent three independent experiments carried out in duplicate. Stars indicate significance where P = <0.01 in T-Test analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835223&req=5

ppat.1005536.g003: The effect of Wolbachia on SFV replicon and transreplicase activity.(A, B) Jw18Wol and Jw18Free cells were transfected with in vitro transcribed SFV1(3F)RLuc-SG-FFLuc RNA, and both RLuc (A) and FFLuc (B) activity was measured 24 h post transfection (hpt). Graphs indicate mean fold change of measurements of luciferase activity where activity in Jw18Wol cells is taken as 1 and represent three independent experiments carried out in triplicate. RLuc activity represents translation of genome RNAs whereas FFLuc indicates translation of the subgenomic mRNA. Error bars represent standard error of mean. Stars indicate significance where P = <0.05 in T-Test analysis. (C, D) Jw18Wol and Jw18Free cells were transfected with two plasmids: one expressing SFV replicase proteins (wt or mutant, under the control of the D. melanogaster Actin promoter) and one expressing viral template and both FFluc (C) and Gluc (D) activity were measured 24 hpt. FFLuc activity represents translation of RNA produced from the Actin promoter (and to some extent the genomic promoter) and Gluc activity represents translation of RNA produced from the subgenomic promoter following replication. Graphs represent relative luciferase activity and represent three independent experiments carried out in duplicate. Stars indicate significance where P = <0.01 in T-Test analysis.
Mentions: As Wolbachia inhibits viral infection or subsequent processes as early as 7 hpi it could be hypothesised that entry of the virus into cells is inhibited, leading to a significant reduction in the number of subsequent replication complexes. In order to test this hypothesis we bypassed viral entry and analysed early translation and replication by transfection of in vitro transcribed capped SFV1(3F)RLuc-SG-FFLuc replicon RNA (Fig 1B). In this SFV-derived replicon RNA, an open reading frame (ORF1) encoding RLuc is fused to the non-structural nsP3 and the second, structural ORF(2) has been deleted and replaced with the FFLuc ORF (see Materials and Methods). Alphavirus gene expression occurs in separate phases which are linked to replicative processes. Initially ORF1 is translated from the RNA genome giving rise to the nsP proteins, which carry out replicative functions. Then a switch from translation to replication occurs leading to production of a full length antisense copy of the genome, the antigenome. Antisense RNA (which most likely exists in a duplex with the original positive-strand genome) is used as a template for synthesis of new genomes; in addition it carries an internal promoter sequence that directs transcription of a subgenomic mRNA encoding the structural proteins (Fig 1) [39]. Thus, expression of structural proteins (or FFLuc marker, Fig 1B) takes place only from these subgenomic RNAs i.e. is fully dependent on the replication process. In contrast, RLuc can be produced both by directly translating the replicon that was transfected into the cells as well as by translating new full-length positive strands, generated during RNA replication. Furthermore, in the absence of structural proteins no new virus particles can be formed preventing the spread of infection. It was found that Wolbachia results in a significant inhibition of early translation and/or replication independent of normal viral entry, with both RLuc and FFLuc readouts being significantly lower in Jw18Wol cells compared to Jw18Free cells (Fig 3A and 3B). We cannot rule out that Wolbachia may also have an effect on entry which we do not observe in these assays.

Bottom Line: Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA.Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity.Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway.

View Article: PubMed Central - PubMed

Affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

ABSTRACT
The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.

Show MeSH
Related in: MedlinePlus