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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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Electron microscopy analysis shows defects in MdBV morphogenesis after vlf-1 and vp39 knockdown.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Image of calyx fluid in the lumen of the reproductive tract of a newly emerged adult female pretreated with ds-eGFP. Each virion consists of one electron dense nucleocapsid (arrowhead) surrounded by a single elongate envelope (arrow). (B) Image of calyx fluid from a wasp pretreated with ds-vlf-1. Note the lower density of virions present relative to (A). (C) Image of calyx fluid from a wasp pretreated with ds-vp39. Scale bars in A-C equal 500 nm. (D) Graph showing that virion density in calyx fluid from wasps treated with ds-vlf-1 was significantly lower than in wasps treated with ds-eGFP or ds-vp39. (E) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-eGFP. The left side of the image shows a large array of assembled MdBV virions. Virions in the process of assembly are visible in the lower right of the image. The insert in the upper right shows virion assembly at higher magnification. Note that assembled virions (*), de novo forming envelopes, empty capsids (arrowhead) and electron dense nucleocapsids in the process of being surrounded by envelopes (arrow). (F) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vlf-1. Unlike normal calyx cells, few virions are assembled into arrays (*). Most of these virions have envelopes that are rounded. Numerous electron dense nucleocapsids without envelopes (arrows) and rounded envelopes without electron dense nucleocapsids are visible. The insert in the upper right shows these defects at higher magnification. It also shows that some rounded envelopes contain empty capsids (arrow). (G) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vp39. Arrays of assembled virions are visible but they are much smaller than the arrays observed in calyx cell nuclei from control wasps. The insert in the upper right shows virions with elongate envelope and electron dense nucleocapsid similar to virions from control wasps. Scale bar for images and inset images E–G equal 500 nm.
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ppat-1003348-g005: Electron microscopy analysis shows defects in MdBV morphogenesis after vlf-1 and vp39 knockdown.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Image of calyx fluid in the lumen of the reproductive tract of a newly emerged adult female pretreated with ds-eGFP. Each virion consists of one electron dense nucleocapsid (arrowhead) surrounded by a single elongate envelope (arrow). (B) Image of calyx fluid from a wasp pretreated with ds-vlf-1. Note the lower density of virions present relative to (A). (C) Image of calyx fluid from a wasp pretreated with ds-vp39. Scale bars in A-C equal 500 nm. (D) Graph showing that virion density in calyx fluid from wasps treated with ds-vlf-1 was significantly lower than in wasps treated with ds-eGFP or ds-vp39. (E) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-eGFP. The left side of the image shows a large array of assembled MdBV virions. Virions in the process of assembly are visible in the lower right of the image. The insert in the upper right shows virion assembly at higher magnification. Note that assembled virions (*), de novo forming envelopes, empty capsids (arrowhead) and electron dense nucleocapsids in the process of being surrounded by envelopes (arrow). (F) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vlf-1. Unlike normal calyx cells, few virions are assembled into arrays (*). Most of these virions have envelopes that are rounded. Numerous electron dense nucleocapsids without envelopes (arrows) and rounded envelopes without electron dense nucleocapsids are visible. The insert in the upper right shows these defects at higher magnification. It also shows that some rounded envelopes contain empty capsids (arrow). (G) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vp39. Arrays of assembled virions are visible but they are much smaller than the arrays observed in calyx cell nuclei from control wasps. The insert in the upper right shows virions with elongate envelope and electron dense nucleocapsid similar to virions from control wasps. Scale bar for images and inset images E–G equal 500 nm.

Mentions: These results could be explained by vp39 and vlf-1 knockdown either adversely affecting virion formation, which would result in calyx fluid containing a lower titer of virus, or causing structural defects that do not reduce virion density but nonetheless compromise function. We therefore examined virion morphology in calyx fluid by transmission electron microscopy (TEM). We previously documented that MdBV virions in calyx fluid consist of a single barrel-shaped nucleocapsid surrounded by a highly elongate envelope [29], [31]. By counting the number of virions in randomly selected fields of view from treatment and control wasp sections, we determined that calyx fluid from a vlf-1 knockdown wasp contained a slightly lower concentration of virions than observed in a control wasp, whereas a vp39 knockdown wasp did not (Figure 5A–D).


Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

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

Electron microscopy analysis shows defects in MdBV morphogenesis after vlf-1 and vp39 knockdown.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Image of calyx fluid in the lumen of the reproductive tract of a newly emerged adult female pretreated with ds-eGFP. Each virion consists of one electron dense nucleocapsid (arrowhead) surrounded by a single elongate envelope (arrow). (B) Image of calyx fluid from a wasp pretreated with ds-vlf-1. Note the lower density of virions present relative to (A). (C) Image of calyx fluid from a wasp pretreated with ds-vp39. Scale bars in A-C equal 500 nm. (D) Graph showing that virion density in calyx fluid from wasps treated with ds-vlf-1 was significantly lower than in wasps treated with ds-eGFP or ds-vp39. (E) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-eGFP. The left side of the image shows a large array of assembled MdBV virions. Virions in the process of assembly are visible in the lower right of the image. The insert in the upper right shows virion assembly at higher magnification. Note that assembled virions (*), de novo forming envelopes, empty capsids (arrowhead) and electron dense nucleocapsids in the process of being surrounded by envelopes (arrow). (F) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vlf-1. Unlike normal calyx cells, few virions are assembled into arrays (*). Most of these virions have envelopes that are rounded. Numerous electron dense nucleocapsids without envelopes (arrows) and rounded envelopes without electron dense nucleocapsids are visible. The insert in the upper right shows these defects at higher magnification. It also shows that some rounded envelopes contain empty capsids (arrow). (G) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vp39. Arrays of assembled virions are visible but they are much smaller than the arrays observed in calyx cell nuclei from control wasps. The insert in the upper right shows virions with elongate envelope and electron dense nucleocapsid similar to virions from control wasps. Scale bar for images and inset images E–G equal 500 nm.
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Related In: Results  -  Collection

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ppat-1003348-g005: Electron microscopy analysis shows defects in MdBV morphogenesis after vlf-1 and vp39 knockdown.M. demolitor larvae were injected with ds-eGFP, ds-vp39 or ds-vlf-1 as shown in Figure 2. (A) Image of calyx fluid in the lumen of the reproductive tract of a newly emerged adult female pretreated with ds-eGFP. Each virion consists of one electron dense nucleocapsid (arrowhead) surrounded by a single elongate envelope (arrow). (B) Image of calyx fluid from a wasp pretreated with ds-vlf-1. Note the lower density of virions present relative to (A). (C) Image of calyx fluid from a wasp pretreated with ds-vp39. Scale bars in A-C equal 500 nm. (D) Graph showing that virion density in calyx fluid from wasps treated with ds-vlf-1 was significantly lower than in wasps treated with ds-eGFP or ds-vp39. (E) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-eGFP. The left side of the image shows a large array of assembled MdBV virions. Virions in the process of assembly are visible in the lower right of the image. The insert in the upper right shows virion assembly at higher magnification. Note that assembled virions (*), de novo forming envelopes, empty capsids (arrowhead) and electron dense nucleocapsids in the process of being surrounded by envelopes (arrow). (F) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vlf-1. Unlike normal calyx cells, few virions are assembled into arrays (*). Most of these virions have envelopes that are rounded. Numerous electron dense nucleocapsids without envelopes (arrows) and rounded envelopes without electron dense nucleocapsids are visible. The insert in the upper right shows these defects at higher magnification. It also shows that some rounded envelopes contain empty capsids (arrow). (G) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-vp39. Arrays of assembled virions are visible but they are much smaller than the arrays observed in calyx cell nuclei from control wasps. The insert in the upper right shows virions with elongate envelope and electron dense nucleocapsid similar to virions from control wasps. Scale bar for images and inset images E–G equal 500 nm.
Mentions: These results could be explained by vp39 and vlf-1 knockdown either adversely affecting virion formation, which would result in calyx fluid containing a lower titer of virus, or causing structural defects that do not reduce virion density but nonetheless compromise function. We therefore examined virion morphology in calyx fluid by transmission electron microscopy (TEM). We previously documented that MdBV virions in calyx fluid consist of a single barrel-shaped nucleocapsid surrounded by a highly elongate envelope [29], [31]. By counting the number of virions in randomly selected fields of view from treatment and control wasp sections, we determined that calyx fluid from a vlf-1 knockdown wasp contained a slightly lower concentration of virions than observed in a control wasp, whereas a vp39 knockdown wasp did not (Figure 5A–D).

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