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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 vp39 and vlf-1 increases DNAse sensitivity and reduces infectivity.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vp39. (B) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vlf-1. (C) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (D) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. (E) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (F) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
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ppat-1003348-g004: Knockdown of vp39 and vlf-1 increases DNAse sensitivity and reduces infectivity.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vp39. (B) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vlf-1. (C) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (D) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. (E) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (F) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.

Mentions: In baculoviruses, VP39 is a major capsid protein while VLF-1 is a structural component, and is also functionally required for capsid production and very late gene expression [24], [40]–[42]. After knocking down vp39 and vlf-1 in M. demolitor (Figure 2C, D), we first assessed whether either treatment affected virion structural integrity by measuring the DNase sensitivity of packaged genomic DNAs as described above. These assays indicated that the abundance of DNase-protected segment B declined by 83% and 78% in vp39 and vlf-1 knockdown samples respectively relative to the control (Figure 4A, B). We also used the non-overlapping dsRNA, ds-vlf-1-2 in these assays, which produced the same result as ds-vlf-1 (Figure S2C).


Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

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

Knockdown of vp39 and vlf-1 increases DNAse sensitivity and reduces infectivity.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vp39. (B) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vlf-1. (C) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (D) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. (E) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (F) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
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ppat-1003348-g004: Knockdown of vp39 and vlf-1 increases DNAse sensitivity and reduces infectivity.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vp39. (B) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged M. demolitor adult females pretreated with ds-eGFP or ds-vlf-1. (C) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (D) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. (E) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vp39. (F) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-eGFP or ds-vlf-1. Error bars, N values, and statistical significance are indicated as defined in Figure 2.
Mentions: In baculoviruses, VP39 is a major capsid protein while VLF-1 is a structural component, and is also functionally required for capsid production and very late gene expression [24], [40]–[42]. After knocking down vp39 and vlf-1 in M. demolitor (Figure 2C, D), we first assessed whether either treatment affected virion structural integrity by measuring the DNase sensitivity of packaged genomic DNAs as described above. These assays indicated that the abundance of DNase-protected segment B declined by 83% and 78% in vp39 and vlf-1 knockdown samples respectively relative to the control (Figure 4A, B). We also used the non-overlapping dsRNA, ds-vlf-1-2 in these assays, which produced the same result as ds-vlf-1 (Figure S2C).

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