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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 the distrubution of virus-derived 21nt interfering RNAs in Jw18 cells.Distribution of 21 nt long viRNAs along sense (upper bars, black; 5’-3’ orientation) or antisense (lower bars, red; 3′-5′ orientation) strands of the SFV genome in the absence (A) (Jw18Free) or presence (B) (Jw18Wol) of Wolbachia. A zoomed in version of (B) is shown in (C) for ease of comparison. Data are from 24 hpi with SFV4(3H)-RLuc, of Jw18 D. melanogaster cells. Concatenated data from 5 independent infections are shown in all panels.
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ppat.1005536.g005: The effect of Wolbachia on the distrubution of virus-derived 21nt interfering RNAs in Jw18 cells.Distribution of 21 nt long viRNAs along sense (upper bars, black; 5’-3’ orientation) or antisense (lower bars, red; 3′-5′ orientation) strands of the SFV genome in the absence (A) (Jw18Free) or presence (B) (Jw18Wol) of Wolbachia. A zoomed in version of (B) is shown in (C) for ease of comparison. Data are from 24 hpi with SFV4(3H)-RLuc, of Jw18 D. melanogaster cells. Concatenated data from 5 independent infections are shown in all panels.

Mentions: A major immune pathway in insects to fight viral infections is the exogenous siRNA pathway which involves the production of virus-derived small interfering RNAs (viRNAs) by the enzyme Dicer-2 acting on virus-derived dsRNA (such as viral replication intermediates) as a substrate [26, 27, 41]. The hallmark of this pathway in insects is the production of 21 nucleotide (nt) viRNAs, a process that has been described in detail for alphavirus infection of mosquitoes and mosquito cells [36, 37, 42]. To test whether the levels of viRNAs were affected by Wolbachia we used high-throughput sequencing of 18-33nt small RNAs from Jw18 cells 24hpi with SFV4(3H)-RLuc. In Jw18Free cells, small RNAs that were 21 nt long and map to the SFV genome were strongly induced upon viral infection (Wilcoxon unpaired test: P = 0.008, Fig 4A, S1A Fig for uninfected controls). 21 nt RNAs mapped equally in both sense and antisense orientations to the viral genome (Fig 4A; P > 0.1 Chi-squared test against a uniform distribution). The length and lack of strand bias or first nucleotide bias (Fig 4A) suggest that these small RNAs are generated by the activity of Dicer-2 on virus-derived dsRNA, probably replication intermediates; moreover 21 nt viRNAs were distributed across the viral genome as previously reported for SFV (Fig 5A) [37, 43] and also other arboviruses [26, 27]. Indeed there was no nucleotide bias seen at any position either in the Jw18Free or Jw18Wol cells (S2A and S2B Fig).


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 the distrubution of virus-derived 21nt interfering RNAs in Jw18 cells.Distribution of 21 nt long viRNAs along sense (upper bars, black; 5’-3’ orientation) or antisense (lower bars, red; 3′-5′ orientation) strands of the SFV genome in the absence (A) (Jw18Free) or presence (B) (Jw18Wol) of Wolbachia. A zoomed in version of (B) is shown in (C) for ease of comparison. Data are from 24 hpi with SFV4(3H)-RLuc, of Jw18 D. melanogaster cells. Concatenated data from 5 independent infections are shown in all panels.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005536.g005: The effect of Wolbachia on the distrubution of virus-derived 21nt interfering RNAs in Jw18 cells.Distribution of 21 nt long viRNAs along sense (upper bars, black; 5’-3’ orientation) or antisense (lower bars, red; 3′-5′ orientation) strands of the SFV genome in the absence (A) (Jw18Free) or presence (B) (Jw18Wol) of Wolbachia. A zoomed in version of (B) is shown in (C) for ease of comparison. Data are from 24 hpi with SFV4(3H)-RLuc, of Jw18 D. melanogaster cells. Concatenated data from 5 independent infections are shown in all panels.
Mentions: A major immune pathway in insects to fight viral infections is the exogenous siRNA pathway which involves the production of virus-derived small interfering RNAs (viRNAs) by the enzyme Dicer-2 acting on virus-derived dsRNA (such as viral replication intermediates) as a substrate [26, 27, 41]. The hallmark of this pathway in insects is the production of 21 nucleotide (nt) viRNAs, a process that has been described in detail for alphavirus infection of mosquitoes and mosquito cells [36, 37, 42]. To test whether the levels of viRNAs were affected by Wolbachia we used high-throughput sequencing of 18-33nt small RNAs from Jw18 cells 24hpi with SFV4(3H)-RLuc. In Jw18Free cells, small RNAs that were 21 nt long and map to the SFV genome were strongly induced upon viral infection (Wilcoxon unpaired test: P = 0.008, Fig 4A, S1A Fig for uninfected controls). 21 nt RNAs mapped equally in both sense and antisense orientations to the viral genome (Fig 4A; P > 0.1 Chi-squared test against a uniform distribution). The length and lack of strand bias or first nucleotide bias (Fig 4A) suggest that these small RNAs are generated by the activity of Dicer-2 on virus-derived dsRNA, probably replication intermediates; moreover 21 nt viRNAs were distributed across the viral genome as previously reported for SFV (Fig 5A) [37, 43] and also other arboviruses [26, 27]. Indeed there was no nucleotide bias seen at any position either in the Jw18Free or Jw18Wol cells (S2A and S2B Fig).

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