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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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Knockdown of p74 and pif-1 reduces the fraction of host cells that express GLC1.8.M. demolitor larvae were injected with ds-eGFP, ds-p74 or ds-pif-1 as shown in Figure 2. (A) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. (B) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
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ppat-1003348-g006: Knockdown of p74 and pif-1 reduces the fraction of host cells that express GLC1.8.M. demolitor larvae were injected with ds-eGFP, ds-p74 or ds-pif-1 as shown in Figure 2. (A) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. (B) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.

Mentions: p74 and the pif genes are known as per os infectivity factors because their loss in baculoviruses such as AcMNPV disables oral infection of host insects by occlusion derived virus [47]–[49]. Each is also a component of the occlusion derived virus envelope where they form a complex with one another [50]. Unlike baculoviruses, BV virions never infect host insects orally but instead are injected into the hemocoel by wasps where they bind to host cells such as hemocytes via fusion of the envelope with the plasma membrane [51]. Nucleocapsids then travel through the cytoplasm to nuclear pores where they release their DNA into the nucleus to initiate transcription of virulence genes like glc1.8[51], [52]. Given the differences in the known functions of per os infectivity factors in baculoviruses relative to the biology of BVs we asked whether the p74 and pif-1-like genes of MdBV still play a role in infectivity by knocking down each (Figure 2E, F) and then conducting the same assays in CiE1 cells as described above. Our results revealed no differences between knockdown and control wasps in the copy number of DNA segment B in CiE1 cells at 24 h post-infection (Figure 6A). However, the fraction of CiE1 cells expressing GLC1.8 on their surface was dramatically lower using virus from p74 and pif-1 knockdown wasps (Figure 6B).


Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

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

Knockdown of p74 and pif-1 reduces the fraction of host cells that express GLC1.8.M. demolitor larvae were injected with ds-eGFP, ds-p74 or ds-pif-1 as shown in Figure 2. (A) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. (B) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
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getmorefigures.php?uid=PMC3649998&req=5

ppat-1003348-g006: Knockdown of p74 and pif-1 reduces the fraction of host cells that express GLC1.8.M. demolitor larvae were injected with ds-eGFP, ds-p74 or ds-pif-1 as shown in Figure 2. (A) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. (B) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP, ds-p74, or ds-pif-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
Mentions: p74 and the pif genes are known as per os infectivity factors because their loss in baculoviruses such as AcMNPV disables oral infection of host insects by occlusion derived virus [47]–[49]. Each is also a component of the occlusion derived virus envelope where they form a complex with one another [50]. Unlike baculoviruses, BV virions never infect host insects orally but instead are injected into the hemocoel by wasps where they bind to host cells such as hemocytes via fusion of the envelope with the plasma membrane [51]. Nucleocapsids then travel through the cytoplasm to nuclear pores where they release their DNA into the nucleus to initiate transcription of virulence genes like glc1.8[51], [52]. Given the differences in the known functions of per os infectivity factors in baculoviruses relative to the biology of BVs we asked whether the p74 and pif-1-like genes of MdBV still play a role in infectivity by knocking down each (Figure 2E, F) and then conducting the same assays in CiE1 cells as described above. Our results revealed no differences between knockdown and control wasps in the copy number of DNA segment B in CiE1 cells at 24 h post-infection (Figure 6A). However, the fraction of CiE1 cells expressing GLC1.8 on their surface was dramatically lower using virus from p74 and pif-1 knockdown wasps (Figure 6B).

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