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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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Analysis of cell growth and infection dynamics of Jw18Wol and Jw18Free cells infected with SFV.(A) Jw18Free cells were infected with SFV(3H)-RLuc at an MOI of 20. Cells were lysed 4, 8, 12 and 24 hpi and RLuc activity determined. The figure represents data from three independent experiments, where each treatment was carried out in triplicate. (B) The density of Jw18Wol and Jw18Free cells 7 and 24 hpi with SFV(3H)-RLuc at an MOI of 20; 0 (seed) is 24 h prior to infection. Data represents three independent experiments carried out in duplicate. (C) Jw18Wol and Jw18Free cells were infected at an MOI of 20 with SFV4(3H)-RLuc and RLuc activity was measured at 7 and 24 hpi. The graph indicates the mean ratio of RLuc activity in Jw18Wol and Jw18Free cells, where Jw18Wol at 7hpi and 24 hpi is equal to one. The data represents five independent experiments carried out in duplicate. Error bars represent the standard error of mean in all figures. Stars indicate significance P = <0.05 in T-Test analysis.
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ppat.1005536.g002: Analysis of cell growth and infection dynamics of Jw18Wol and Jw18Free cells infected with SFV.(A) Jw18Free cells were infected with SFV(3H)-RLuc at an MOI of 20. Cells were lysed 4, 8, 12 and 24 hpi and RLuc activity determined. The figure represents data from three independent experiments, where each treatment was carried out in triplicate. (B) The density of Jw18Wol and Jw18Free cells 7 and 24 hpi with SFV(3H)-RLuc at an MOI of 20; 0 (seed) is 24 h prior to infection. Data represents three independent experiments carried out in duplicate. (C) Jw18Wol and Jw18Free cells were infected at an MOI of 20 with SFV4(3H)-RLuc and RLuc activity was measured at 7 and 24 hpi. The graph indicates the mean ratio of RLuc activity in Jw18Wol and Jw18Free cells, where Jw18Wol at 7hpi and 24 hpi is equal to one. The data represents five independent experiments carried out in duplicate. Error bars represent the standard error of mean in all figures. Stars indicate significance P = <0.05 in T-Test analysis.

Mentions: The cells were first antibiotic treated to generate a Wolbachia-free control cell line, which we refer to as Jw18Free cells. These were infected at a multiplicity of infection (MOI) of 20 and cells were lysed 4, 8, 12 and 24 hours post infection (hpi) and RLuc activity measured. Over a 24 hour period RLuc activity gradually increased, indicating that SFV4(3H)-RLuc can infect and replicate in Jw18Free cells (Fig 2A). In order to rule out any effect of Wolbachia or SFV on cell growth we next compared the growth of cells Jw18Free and Jw18Wol cells either infected or not infected with SFV4(3H)-RLuc. There was no significant difference between any of the treatments observed, indicating that neither Wolbachia nor SFV4(3H)-RLuc infection affected cell growth (Fig 2B). It is important to note that SFV does not cause cytopathic effects in insect cells and therefore cells are able to continue to grow even under high infection rates.


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)

Analysis of cell growth and infection dynamics of Jw18Wol and Jw18Free cells infected with SFV.(A) Jw18Free cells were infected with SFV(3H)-RLuc at an MOI of 20. Cells were lysed 4, 8, 12 and 24 hpi and RLuc activity determined. The figure represents data from three independent experiments, where each treatment was carried out in triplicate. (B) The density of Jw18Wol and Jw18Free cells 7 and 24 hpi with SFV(3H)-RLuc at an MOI of 20; 0 (seed) is 24 h prior to infection. Data represents three independent experiments carried out in duplicate. (C) Jw18Wol and Jw18Free cells were infected at an MOI of 20 with SFV4(3H)-RLuc and RLuc activity was measured at 7 and 24 hpi. The graph indicates the mean ratio of RLuc activity in Jw18Wol and Jw18Free cells, where Jw18Wol at 7hpi and 24 hpi is equal to one. The data represents five independent experiments carried out in duplicate. Error bars represent the standard error of mean in all figures. Stars indicate significance P = <0.05 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.g002: Analysis of cell growth and infection dynamics of Jw18Wol and Jw18Free cells infected with SFV.(A) Jw18Free cells were infected with SFV(3H)-RLuc at an MOI of 20. Cells were lysed 4, 8, 12 and 24 hpi and RLuc activity determined. The figure represents data from three independent experiments, where each treatment was carried out in triplicate. (B) The density of Jw18Wol and Jw18Free cells 7 and 24 hpi with SFV(3H)-RLuc at an MOI of 20; 0 (seed) is 24 h prior to infection. Data represents three independent experiments carried out in duplicate. (C) Jw18Wol and Jw18Free cells were infected at an MOI of 20 with SFV4(3H)-RLuc and RLuc activity was measured at 7 and 24 hpi. The graph indicates the mean ratio of RLuc activity in Jw18Wol and Jw18Free cells, where Jw18Wol at 7hpi and 24 hpi is equal to one. The data represents five independent experiments carried out in duplicate. Error bars represent the standard error of mean in all figures. Stars indicate significance P = <0.05 in T-Test analysis.
Mentions: The cells were first antibiotic treated to generate a Wolbachia-free control cell line, which we refer to as Jw18Free cells. These were infected at a multiplicity of infection (MOI) of 20 and cells were lysed 4, 8, 12 and 24 hours post infection (hpi) and RLuc activity measured. Over a 24 hour period RLuc activity gradually increased, indicating that SFV4(3H)-RLuc can infect and replicate in Jw18Free cells (Fig 2A). In order to rule out any effect of Wolbachia or SFV on cell growth we next compared the growth of cells Jw18Free and Jw18Wol cells either infected or not infected with SFV4(3H)-RLuc. There was no significant difference between any of the treatments observed, indicating that neither Wolbachia nor SFV4(3H)-RLuc infection affected cell growth (Fig 2B). It is important to note that SFV does not cause cytopathic effects in insect cells and therefore cells are able to continue to grow even under high infection rates.

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