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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 changes in Wolbachia and D. melanogaster gene expression upon viral infection.Jw18Wol cells infected with SFV4(3H)-RLuc were analyzed. (A) Sequence coverage of Wolbachia genes. Genes are arranged from lowest to highest coverage on the X axis. In the legend + refers to the presence of SFV. (B-C) The response to infection (log2 fold expression change) for each Wolbachia gene is shown against expression level (average log2 count per million [CPM]) at (B) 7 h and (C) 24 h after infection. (D-E) The response to infection of D. melanogaster genes. Genes that significantly change in expression at 10% false discovery rate (FDR) are shown in red (there were no significant induction/repression for Wolbachia genes).
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ppat.1005536.g007: Analysis of changes in Wolbachia and D. melanogaster gene expression upon viral infection.Jw18Wol cells infected with SFV4(3H)-RLuc were analyzed. (A) Sequence coverage of Wolbachia genes. Genes are arranged from lowest to highest coverage on the X axis. In the legend + refers to the presence of SFV. (B-C) The response to infection (log2 fold expression change) for each Wolbachia gene is shown against expression level (average log2 count per million [CPM]) at (B) 7 h and (C) 24 h after infection. (D-E) The response to infection of D. melanogaster genes. Genes that significantly change in expression at 10% false discovery rate (FDR) are shown in red (there were no significant induction/repression for Wolbachia genes).

Mentions: To investigate whether Wolbachia itself might mount an active antiviral response after infection, we tested if there is a transcriptional response of Wolbachia to viral infection. We sequenced total RNA from Jw18Wol cells 7 and 24 hpi with SFV4(3H)-RLuc virus together with uninfected controls. Over 229 million reads could be mapped to the D. melanogaster, Wolbachia or SFV transcriptomes (excluding D. melanogaster rRNA). Of these 85.6% mapped to D. melanogaster and 12.4% mapped to Wolbachia. In the virus-infected cells, 3.8% of reads mapped to the SFV genome, and this dropped to 0.03% for cells that were not challenged with the virus. No Wolbachia genes were differentially expressed in response to viral infection at either time point (Fig 7B and 7C). There are three reasons to believe that this is a true lack of a transcriptional response and not simply a lack of statistical power. First, across many transcripts we were able to detect a transcriptional response of the cells to SFV in the same experiment (Fig 7D and 7E), and the coverage of many of these differentially expressed transcripts was lower than for many Wolbachia transcripts (Fig 7B–7E). Second, we had a very large dataset. In each of the 4 treatments about two thirds of the Wolbachia transcripts had over 100 reads mapped to them (Fig 7A), which is expected to give good power to detect differential expression. Compared to most published RNAseq experiments ours was a highly replicated experiment involving 40 libraries (biological replicates) across 4 lanes of Illumina HiSeq. Third, if genes with very low-expression are ignored, our estimates of Wolbachia gene expression levels in cells with and without SFV were nearly identical (Fig 7B and 7C, log2FC≈0). Therefore, the lack of differential expression cannot be attributed to a lack of statistical power.


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 changes in Wolbachia and D. melanogaster gene expression upon viral infection.Jw18Wol cells infected with SFV4(3H)-RLuc were analyzed. (A) Sequence coverage of Wolbachia genes. Genes are arranged from lowest to highest coverage on the X axis. In the legend + refers to the presence of SFV. (B-C) The response to infection (log2 fold expression change) for each Wolbachia gene is shown against expression level (average log2 count per million [CPM]) at (B) 7 h and (C) 24 h after infection. (D-E) The response to infection of D. melanogaster genes. Genes that significantly change in expression at 10% false discovery rate (FDR) are shown in red (there were no significant induction/repression for Wolbachia genes).
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getmorefigures.php?uid=PMC4835223&req=5

ppat.1005536.g007: Analysis of changes in Wolbachia and D. melanogaster gene expression upon viral infection.Jw18Wol cells infected with SFV4(3H)-RLuc were analyzed. (A) Sequence coverage of Wolbachia genes. Genes are arranged from lowest to highest coverage on the X axis. In the legend + refers to the presence of SFV. (B-C) The response to infection (log2 fold expression change) for each Wolbachia gene is shown against expression level (average log2 count per million [CPM]) at (B) 7 h and (C) 24 h after infection. (D-E) The response to infection of D. melanogaster genes. Genes that significantly change in expression at 10% false discovery rate (FDR) are shown in red (there were no significant induction/repression for Wolbachia genes).
Mentions: To investigate whether Wolbachia itself might mount an active antiviral response after infection, we tested if there is a transcriptional response of Wolbachia to viral infection. We sequenced total RNA from Jw18Wol cells 7 and 24 hpi with SFV4(3H)-RLuc virus together with uninfected controls. Over 229 million reads could be mapped to the D. melanogaster, Wolbachia or SFV transcriptomes (excluding D. melanogaster rRNA). Of these 85.6% mapped to D. melanogaster and 12.4% mapped to Wolbachia. In the virus-infected cells, 3.8% of reads mapped to the SFV genome, and this dropped to 0.03% for cells that were not challenged with the virus. No Wolbachia genes were differentially expressed in response to viral infection at either time point (Fig 7B and 7C). There are three reasons to believe that this is a true lack of a transcriptional response and not simply a lack of statistical power. First, across many transcripts we were able to detect a transcriptional response of the cells to SFV in the same experiment (Fig 7D and 7E), and the coverage of many of these differentially expressed transcripts was lower than for many Wolbachia transcripts (Fig 7B–7E). Second, we had a very large dataset. In each of the 4 treatments about two thirds of the Wolbachia transcripts had over 100 reads mapped to them (Fig 7A), which is expected to give good power to detect differential expression. Compared to most published RNAseq experiments ours was a highly replicated experiment involving 40 libraries (biological replicates) across 4 lanes of Illumina HiSeq. Third, if genes with very low-expression are ignored, our estimates of Wolbachia gene expression levels in cells with and without SFV were nearly identical (Fig 7B and 7C, log2FC≈0). Therefore, the lack of differential expression cannot be attributed to a lack of statistical power.

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