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Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

Burke GR, Thomas SA, Eum JH, Strand MR - PLoS Pathog. (2013)

Bottom Line: Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses.We further show that RNA interference effectively and specifically knocks down MdBV gene expression.Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of Georgia, Athens, Georgia, United States of America. grburke@uga.edu

ABSTRACT
Viruses are usually thought to form parasitic associations with hosts, but all members of the family Polydnaviridae are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from Microplitis demolitor bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (lef-4, lef-9), capsid assembly (vp39, vlf-1), and envelope formation (p74, pif-1) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions. Additional experiments suggested that vlf-1 together with the nudivirus-like gene int-1 also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation.

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RNAi knockdown of six MdBV putative replication genes.M. demolitor larvae were injected with double-stranded RNA (dsRNA) specific for (A) lef-4, (B) lef-9, (C) vp39, (D) vlf-1, (E) p74, (F) pif-1, (G) int-1. Control larvae were injected with ds-eGFP. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated. The bars in each graph compare copy number of each target gene per ng of total RNA in wasps treated with dsRNA specific for the target gene versus ds-eGFP (control). Error bars represent one standard error from the mean. The number of individuals examined for each treatment is indicated by the N value at the bottom of each bar. Statistical significance is indicated by asterisks: *, p<0.05; **, p<0.001; ***, p<0.0001; N. S., not significant. (H) Knockdown of lef-9 depletes LEF-9 protein. M. demolitor larvae were injected with ds-lef-9 as in (B). Total protein from the ovaries of a newly emerged adult wasp was loaded into lanes of an SDS-PAGE gel followed by immunoblot analysis using an MdBV anti-LEF-9 antibody (1∶5000). The left lanes show results from 5 individual M. demolitor females treated with ds-lef-9 (knockdown), while the lanes on the right show results from 4 females treated with ds-eGFP (control). The predicted size of MdBV LEF-9 is 74.4 kDa.
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ppat-1003348-g002: RNAi knockdown of six MdBV putative replication genes.M. demolitor larvae were injected with double-stranded RNA (dsRNA) specific for (A) lef-4, (B) lef-9, (C) vp39, (D) vlf-1, (E) p74, (F) pif-1, (G) int-1. Control larvae were injected with ds-eGFP. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated. The bars in each graph compare copy number of each target gene per ng of total RNA in wasps treated with dsRNA specific for the target gene versus ds-eGFP (control). Error bars represent one standard error from the mean. The number of individuals examined for each treatment is indicated by the N value at the bottom of each bar. Statistical significance is indicated by asterisks: *, p<0.05; **, p<0.001; ***, p<0.0001; N. S., not significant. (H) Knockdown of lef-9 depletes LEF-9 protein. M. demolitor larvae were injected with ds-lef-9 as in (B). Total protein from the ovaries of a newly emerged adult wasp was loaded into lanes of an SDS-PAGE gel followed by immunoblot analysis using an MdBV anti-LEF-9 antibody (1∶5000). The left lanes show results from 5 individual M. demolitor females treated with ds-lef-9 (knockdown), while the lanes on the right show results from 4 females treated with ds-eGFP (control). The predicted size of MdBV LEF-9 is 74.4 kDa.

Mentions: The genes we selected reside in the MdBV proviral genome and each is transcribed in ovary calyx cells during replication [7]. However, conventional knock out techniques used to characterize baculovirus gene function are untenable because the DNA domains where these genes reside are not replicated and packaged into MdBV virions. We thus assessed whether RNAi could be used to knock down transcription of these genes in M. demolitor. Since MdBV replication begins in the pupal stage of the wasp, we developed methods for injecting gene-specific dsRNAs into wasp larvae after they emerged from a host caterpillar and spun a cocoon. We then compared the abundance of each targeted transcript in newly emerged adult wasps by qPCR relative to treatment with a non-specific dsRNA (ds-eGFP). Our results showed that we reduced transcript abundance on average 60–99% for each gene we targeted (Figure 2). Using an antibody we generated to MdBV LEF-9, we also confirmed that knockdown at the transcript level resulted in knockdown of the corresponding protein (Figure 2).


Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

Burke GR, Thomas SA, Eum JH, Strand MR - PLoS Pathog. (2013)

RNAi knockdown of six MdBV putative replication genes.M. demolitor larvae were injected with double-stranded RNA (dsRNA) specific for (A) lef-4, (B) lef-9, (C) vp39, (D) vlf-1, (E) p74, (F) pif-1, (G) int-1. Control larvae were injected with ds-eGFP. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated. The bars in each graph compare copy number of each target gene per ng of total RNA in wasps treated with dsRNA specific for the target gene versus ds-eGFP (control). Error bars represent one standard error from the mean. The number of individuals examined for each treatment is indicated by the N value at the bottom of each bar. Statistical significance is indicated by asterisks: *, p<0.05; **, p<0.001; ***, p<0.0001; N. S., not significant. (H) Knockdown of lef-9 depletes LEF-9 protein. M. demolitor larvae were injected with ds-lef-9 as in (B). Total protein from the ovaries of a newly emerged adult wasp was loaded into lanes of an SDS-PAGE gel followed by immunoblot analysis using an MdBV anti-LEF-9 antibody (1∶5000). The left lanes show results from 5 individual M. demolitor females treated with ds-lef-9 (knockdown), while the lanes on the right show results from 4 females treated with ds-eGFP (control). The predicted size of MdBV LEF-9 is 74.4 kDa.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649998&req=5

ppat-1003348-g002: RNAi knockdown of six MdBV putative replication genes.M. demolitor larvae were injected with double-stranded RNA (dsRNA) specific for (A) lef-4, (B) lef-9, (C) vp39, (D) vlf-1, (E) p74, (F) pif-1, (G) int-1. Control larvae were injected with ds-eGFP. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated. The bars in each graph compare copy number of each target gene per ng of total RNA in wasps treated with dsRNA specific for the target gene versus ds-eGFP (control). Error bars represent one standard error from the mean. The number of individuals examined for each treatment is indicated by the N value at the bottom of each bar. Statistical significance is indicated by asterisks: *, p<0.05; **, p<0.001; ***, p<0.0001; N. S., not significant. (H) Knockdown of lef-9 depletes LEF-9 protein. M. demolitor larvae were injected with ds-lef-9 as in (B). Total protein from the ovaries of a newly emerged adult wasp was loaded into lanes of an SDS-PAGE gel followed by immunoblot analysis using an MdBV anti-LEF-9 antibody (1∶5000). The left lanes show results from 5 individual M. demolitor females treated with ds-lef-9 (knockdown), while the lanes on the right show results from 4 females treated with ds-eGFP (control). The predicted size of MdBV LEF-9 is 74.4 kDa.
Mentions: The genes we selected reside in the MdBV proviral genome and each is transcribed in ovary calyx cells during replication [7]. However, conventional knock out techniques used to characterize baculovirus gene function are untenable because the DNA domains where these genes reside are not replicated and packaged into MdBV virions. We thus assessed whether RNAi could be used to knock down transcription of these genes in M. demolitor. Since MdBV replication begins in the pupal stage of the wasp, we developed methods for injecting gene-specific dsRNAs into wasp larvae after they emerged from a host caterpillar and spun a cocoon. We then compared the abundance of each targeted transcript in newly emerged adult wasps by qPCR relative to treatment with a non-specific dsRNA (ds-eGFP). Our results showed that we reduced transcript abundance on average 60–99% for each gene we targeted (Figure 2). Using an antibody we generated to MdBV LEF-9, we also confirmed that knockdown at the transcript level resulted in knockdown of the corresponding protein (Figure 2).

Bottom Line: Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses.We further show that RNA interference effectively and specifically knocks down MdBV gene expression.Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of Georgia, Athens, Georgia, United States of America. grburke@uga.edu

ABSTRACT
Viruses are usually thought to form parasitic associations with hosts, but all members of the family Polydnaviridae are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from Microplitis demolitor bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (lef-4, lef-9), capsid assembly (vp39, vlf-1), and envelope formation (p74, pif-1) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions. Additional experiments suggested that vlf-1 together with the nudivirus-like gene int-1 also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation.

Show MeSH
Related in: MedlinePlus