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1. Functional characterization of Microplitis demolitor bracovirus genes that encode nucleocapsid components

   https://doi.org/10.1128/jvi.00817-23  

   Lorenzi, Ange; Arvin, Michael J; Burke, Gaelen R; Strand, Michael R (November 2023, Journal of Virology)

   Parrish, Colin R (Ed.)

   ABSTRACT Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (order Hymenoptera: Family Braconidae). BVs produce replication-defective virions that adult female wasps use to transfer DNAs encoding virulence genes to parasitized hosts. Some BV genes are shared with nudiviruses and baculoviruses with studies of the latter providing insights on function, whereas other genes are only known from nudiviruses or other BVs which provide no functional insights. A proteomic analysis ofMicroplitis demolitorbracovirus (MdBV) virions recently identified 16 genes encoding nucleocapsid components. In this study, we further characterized most of these genes. Some nucleocapsid genes exhibited early or intermediate expression profiles, while others exhibited late expression profiles. RNA interference (RNAi) assays together with transmission electron microscopy indicatedvp39,HzNVorf9-like2,HzNVorf93-like,HzNVorf106-like,HzNVorf118-like,and 27bare required to produce capsids with a normal barrel-shaped morphology. RNAi knockdown ofvlf-1a,vlf-1b-1,vlf-1b-2,int-1,andp6.9-1did not alter the formation of barrel-shaped capsids but each reduced processing of amplified proviral segments and DNA packaging as evidenced by the formation of electron translucent capsids. All of the genes required for normal capsid assembly were also required for proviral segment processing and DNA packaging. Collectively, our results deorphanize several BV genes with previously unknown roles in virion morphogenesis. IMPORTANCEUnderstanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of theMicroplitis demolitorbracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding proteinp6.9-1are required for DNA processing and packaging into nucleocapsids. 

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2. Roles of Cellular NSF Protein in Entry and Nuclear Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

   https://doi.org/10.1128/jvi.01111-17  

   Guo, Ya; Yue, Qi; Gao, Jinli; Wang, Zhe; Chen, Yun-Ru; Blissard, Gary W.; Liu, Tong-Xian; Li, Zhaofei; Sandri-Goldin, Rozanne M. (September 2017, Journal of Virology)

   ABSTRACT In eukaryotic cells, the s oluble N -ethylmaleimide- s ensitive f actor (NSF) a ttachment protein re ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses. 

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3. The Complete Genome of Chelonus insularis Reveals Dynamic Arrangement of Genome Components in Parasitoid Wasps That Produce Bracoviruses

   https://doi.org/10.1128/JVI.01573-21  

   Mao, Meng; Strand, Michael R.; Burke, Gaelen R. (March 2022, Journal of Virology)

   Parrish, Colin R. (Ed.)

   ABSTRACT Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae ( Microplitis demolitor and Cotesia congregata ) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis , which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized compared to M. demolitor and C. congregata . Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. IMPORTANCE Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This work characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis , which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function. 

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4. Comparative transcriptome analyses of the Drosophila pupal eye

   https://doi.org/10.1093/g3journal/jkaa003  

   DeAngelis, Miles W; Coolon, Joseph D; Johnson, Ruth I (January 2021, G3 Genes|Genomes|Genetics)

   Lott, S (Ed.)

   Abstract Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. Many of the genes required for tissue patterning and organization are highly conserved between phyla. This has led to the emergence of several model organisms and developmental systems that are used to study tissue morphogenesis. One such model is the Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, we dissected wild-type Canton S pupal eyes and performed RNA-sequencing. Our analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. We also identified differential expression of genes not previously implicated in pupal eye morphogenesis such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors. 

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5. Investigating Virus–Host Interactions in Cultured Primary Honey Bee Cells

   https://doi.org/10.3390/insects12070653  

   McMenamin, Alexander J.; Parekh, Fenali; Lawrence, Verena; Flenniken, Michelle L. (July 2021, Insects)

   Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap. 

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