A novel and diverse family of filamentous DNA viruses associated with parasitic wasps.

Large dsDNA viruses from the Naldaviricetes class are currently composed of four viral families infecting insects and/or crustaceans. Since the 1970s, particles described as filamentous viruses (FVs) have been observed by electronic microscopy in several species of Hymenoptera parasitoids but until recently, no genomic data was available. This study provides the first comparative morphological and genomic analysis of these FVs. We analyzed the genomes of seven FVs, six of which were newly obtained, to gain a better understanding of their evolutionary history. We show that these FVs share all genomic features of the Naldaviricetes while encoding five specific core genes that distinguish them from their closest relatives, the Hytrosaviruses. By mining public databases, we show that FVs preferentially infect Hymenoptera with parasitoid lifestyle and that these viruses have been repeatedly integrated into the genome of many insects, particularly Hymenoptera parasitoids, overall suggesting a long-standing specialization of these viruses to parasitic wasps. Finally, we propose a taxonomical revision of the class Naldaviricetes in which FVs related to the Leptopilina boulardi FV constitute a fifth family. We propose to name this new family, Filamentoviridae.

The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.

DNA Polymerase Diversity Reveals Multiple Incursions of Polintons During Nematode Evolution.

Polintons are double-stranded DNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family but encode a distinct protein-primed DNA polymerase B (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting interphylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of an HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

Endoparasitoid lifestyle promotes endogenization and domestication of dsDNA viruses.

The accidental endogenization of viral elements within eukaryotic genomes can occasionally provide significant evolutionary benefits, giving rise to their long-term retention, that is, to viral domestication. For instance, in some endoparasitoid wasps (whose immature stages develop inside their hosts), the membrane-fusion property of double-stranded DNA viruses have been repeatedly domesticated following ancestral endogenizations. The endogenized genes provide female wasps with a delivery tool to inject virulence factors that are essential to the developmental success of their offspring. Because all known cases of viral domestication involve endoparasitic wasps, we hypothesized that this lifestyle, relying on a close interaction between individuals, may have promoted the endogenization and domestication of viruses. By analyzing the composition of 124 Hymenoptera genomes, spread over the diversity of this clade and including free-living, ecto, and endoparasitoid species, we tested this hypothesis. Our analysis first revealed that double-stranded DNA viruses, in comparison with other viral genomic structures (ssDNA, dsRNA, ssRNA), are more often endogenized and domesticated (that is, retained by selection) than expected from their estimated abundance in insect viral communities. Second, our analysis indicates that the rate at which dsDNA viruses are endogenized is higher in endoparasitoids than in ectoparasitoids or free-living hymenopterans, which also translates into more frequent events of domestication. Hence, these results are consistent with the hypothesis that the endoparasitoid lifestyle has facilitated the endogenization of dsDNA viruses, in turn, increasing the opportunities of domestications that now play a central role in the biology of many endoparasitoid lineages.

Multiple Nucleocapsid Structural Forms of Shrimp White Spot Syndrome Virus Suggests a Novel Viral Morphogenetic Pathway.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

Tryptophan Residues Are Critical for Portal Protein Assembly and Incorporation in Bacteriophage P22.

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly.

Genome Plasticity of African Swine Fever Virus: Implications for Diagnostics and Live-Attenuated Vaccines.

African swine fever (ASF) is a highly contagious transboundary viral hemorrhagic disease of domestic and wild pigs presenting a significant threat to the global swine industry. Following its introduction in Caucasus, Georgia, in 2007, the genome of the genotype II of African swine fever virus (ASFV) strain Georgia-07 and its derivatives accumulated significant mutations, resulting in the emergence of genetic variants within short epidemiological timescales as it spreads and infects different hosts in diverse ecosystems, causing outbreaks in Europe, South Asia, South East Asia and the Caribbean. This suggests that ASFV, with a comparatively large and complex DNA genome, is susceptible to genetic mutations including deletions and that although the virus is environmentally stable, it is genetically unstable. This has implications for the development of vaccines and diagnostic tests for disease detection and surveillance. Analysis of the ASFV genome revealed recombination hotspots, which in double-stranded DNA (dsDNA) viruses represent key drivers of genetic diversity. The ability of pox virus, a dsDNA virus with a genome complexity similar to ASFV, regaining virulence following the deletion of a virulence gene via gene amplification, coupled with the recent emergence and spread of live-attenuated ASFV vaccine strains causing disease and death in pigs in China, raise legitimate concerns around the use of live-attenuated ASFV vaccines in non-endemic regions to control the potential introduction. Further research into the risk of using live-attenuated ASFV in non-endemic regions is highly needed.

Assessing the Impact of a Viral Infection on the Expression of Transposable Elements in the Cabbage Looper Moth (Trichoplusia ni).

Most studies of stress-induced transposable element (TE) expression have so far focused on abiotic sources of stress. Here, we analyzed the impact of an infection by the AcMNPV baculovirus on TE expression in a cell line (Tnms42) and midgut tissues of the cabbage looper moth (Trichoplusia ni). We find that a large fraction of TE families (576/636 in Tnms42 cells and 503/612 in midgut) is lowly expressed or not expressed at all [≤ 4 transcripts per million (TPM)] in the uninfected condition (median TPM of 0.37 in Tnms42 and 0.46 in midgut cells). In the infected condition, a total of 62 and 187 TE families were differentially expressed (DE) in midgut and Tnms42 cells, respectively, with more up- (46) than downregulated (16) TE families in the former and as many up- (91) as downregulated (96) TE families in the latter. Expression log2 fold changes of DE TE families varied from -4.95 to 9.11 in Tnms42 cells and from -4.28 to 7.66 in midgut. Large variations in expression profiles of DE TEs were observed depending on the type of cells and on time after infection. Overall, the impact of AcMNPV on TE expression in T. ni is moderate but potentially sufficient to affect TE activity and genome architecture. Interestingly, one host-derived TE integrated into AcMNPV genomes is highly expressed in infected Tnms42 cells. This result shows that virus-borne TEs can be expressed, further suggesting that they may be able to transpose and that viruses may act as vectors of horizontal transfer of TEs in insects.

Antipoxvirus Activity Evaluation of Optimized Corroles Based on Development of Autofluorescent ANCHOR Myxoma Virus.

A series of 43 antiviral corrole-based molecules have been tested on myxoma virus (Lausanne-like T1MYXV strain). An autofluorescent MYXV, with an ANCHOR cassette, has been used for the studies. A2B-fluorocorroles display various toxicities, from 40 being very toxic (CC50 = 1.7 µM) to nontoxic 38 (CC50 > 50 µM), whereas A3-fluorocorroles, with one to three fluorine atoms, are not toxic (with the exception of corroles 9, 10, and 22). In vitro, these compounds show a good selectivity index when used alone. Corrole 35 seems to be the most promising compound, which displays a high selectivity index with the lowest IC50. Interestingly, this "Hit" corrole is easy to synthesize in a two-step reaction. Upscaling production up to 25 g has been carried out for in vivo tests. In vivo studies on New Zealand white rabbits infected with myxoma virus show that symptoms are delayed and animal weight is increased upon treatment, while no acute toxicity of the corrole molecule was detected.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Architecture of the herpesvirus genome-packaging complex and implications for DNA translocation.

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33-unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

Brincidofovir: understanding its unique profile and potential role against adenovirus and other viral infections.

Brincidofovir (BCV) is a lipid conjugate of cidofovir with good oral bioavailability, enabling optimal intracellular levels of the active drug. Lower rates of nephrotoxicity and myelotoxicity make it a favorable alternative. Despite a greater safety profile among pediatric hematopoietic cell transplant recipients, the oral formulation has been associated with increased gastrointestinal toxicity in adult hematopoietic cell transplant recipients. Oral BCV continues to be developed as a countermeasure against smallpox, while a potentially safer intravenous preparation has been out licensed to another company. BCV has demonstrated great in vitro potency against double-stranded DNA viruses, especially adenovirus. Because of its importance for immunocompromised patients, this review aims to evaluate BCV's clinical and safety profile to support its continued development.

Mitochondrial and Innate Immunity Transcriptomes from Spodoptera frugiperda Larvae Infected with the Spodoptera frugiperda Ascovirus.

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.

Architecture of the herpesvirus genome-packaging complex and implications for DNA translocation

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33-unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

Architecture of the herpesvirus genome-packaging complex and implications for DNA translocation

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33-unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

Cytomegalovirus distribution and evolution in hominines.

Herpesviruses are thought to have evolved in very close association with their hosts. This is notably the case for cytomegaloviruses (CMVs; genus Cytomegalovirus) infecting primates, which exhibit a strong signal of co-divergence with their hosts. Some herpesviruses are however known to have crossed species barriers. Based on a limited sampling of CMV diversity in the hominine (African great ape and human) lineage, we hypothesized that chimpanzees and gorillas might have mutually exchanged CMVs in the past. Here, we performed a comprehensive molecular screening of all 9 African great ape species/subspecies, using 675 fecal samples collected from wild animals. We identified CMVs in eight species/subspecies, notably generating the first CMV sequences from bonobos. We used this extended dataset to test competing hypotheses with various degrees of co-divergence/number of host switches while simultaneously estimating the dates of these events in a Bayesian framework. The model best supported by the data involved the transmission of a gorilla CMV to the panine (chimpanzee and bonobo) lineage and the transmission of a panine CMV to the gorilla lineage prior to the divergence of chimpanzees and bonobos, more than 800,000 years ago. Panine CMVs then co-diverged with their hosts. These results add to a growing body of evidence suggesting that viruses with a double-stranded DNA genome (including other herpesviruses, adenoviruses, and papillomaviruses) often jumped between hominine lineages over the last few million years.

In vitro comparison of currently available and investigational antiviral agents against pathogenic human double-stranded DNA viruses: A systematic literature review.

BACKGROUND: Double-stranded (ds) DNA virus infections often occur concomitantly in immunocompromised patients. We performed a systematic search of published in vitro activity for nine approved and investigational antivirals to understand the spectrum of in vitro activity against dsDNA viruses. METHODS: A literature search was performed (PubMed and the WoS Core Collection) using keywords related to: 1) targeted approved/developmental antivirals (acyclovir, artesunate, brincidofovir, cidofovir, cyclopropavir (filociclovir), foscarnet, ganciclovir, letermovir, and maribavir); 2) pathogenic dsDNA viruses; 3) in vitro activity. We summarized data from 210 publications. RESULTS: Activity against ≤3 viruses was documented for maribavir (cytomegalovirus, Epstein-Barr virus), and letermovir, while activity against > 3 viruses was shown for ganciclovir, cidofovir, acyclovir, foscarnet, cyclopropavir, artesunate, and brincidofovir. The EC50 values of brincidofovir were the lowest, ranging from 0.001 to 0.27 µM, for all viruses except papillomaviruses. The next most potent agents included cidofovir, ganciclovir, foscarnet, and acyclovir with EC50 values between 0.1 µM and >10 µM for cytomegalovirus, herpes simplex virus, and adenovirus. CONCLUSION: Most of the identified antivirals had in vitro activity against more than one dsDNA virus. Brincidofovir and cidofovir have broad-spectrum activity, and brincidofovir has the lowest EC50 values. These findings could assist clinical practice and developmental research.

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis.

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Šresolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = ß = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

The N Terminus of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Is Required for Efficient Viral DNA Replication and Virus and Occlusion Body Production.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) plays a crucial role in viral DNA synthesis, and the N terminus (residues 1 to 186) is highly conserved in the baculovirus DNApol family. However, the functional role of the N terminus of DNApol has not yet been characterized. Here we report a functional analysis of the AcMNPV DNApol N terminus. We truncated the DNApol N terminus to construct truncation mutants Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186, which lack 64, 110, and 186 N-terminal residues, respectively. Although the truncation mutants rescued viral DNA synthesis and infectious virus production, the level of DNA replication decreased, and Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186 showed 10-fold, 89-fold, and 891-fold reductions in infectious viral yield compared to that of the wild-type repair virus, respectively. Production of occlusion bodies was compromised for all truncation mutants. Further bioinformatic analysis showed that the first 64 amino acids (aa) at the extreme N terminus contains a conserved α(-helix)-ß(-sheet)-ß-ß secondary-structure region, and further downstream sequence from aa 67 to 186 is comprised of four conserved sequence motifs. Multiple alanine point substitutions in the α-ß-ß-ß structure region or the four sequence motifs in the N terminus impaired viral DNA replication and resulted in reduction of virus yield and occlusion body production. Together, our results suggested that the secondary structure and four conserved motifs within the N terminus of AcMNPV DNApol are important for viral DNA synthesis, infectious virus yield, and production of occlusion bodies.IMPORTANCE DNA polymerase (DNApol) is highly conserved in all baculoviruses and is required for viral DNA replication. The N terminus is one of the highly conserved regions of baculovirus DNApols. Our results showed that the N terminus of baculovirus DNA polymerase plays an important role in efficient viral DNA synthesis and infectious virus yield and production of occlusion bodies. We identified five features, including a highly conserved secondary structure and four conserved amino acid motifs, in the AcMNPV DNApol N terminus, all of which are important for efficient viral DNA synthesis, infectious virus yield, and production of occlusion bodies.

Transcriptome Analysis of the Spodoptera frugiperda Ascovirus In Vivo Provides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny.

Ascoviruses are double-stranded DNA (dsDNA) viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode a caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific transcriptome sequencing (RNA-Seq) to study transcription in vivo in third instars of Spodoptera frugiperda infected with the type species, Spodoptera frugiperda ascovirus1a (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes: early, late, and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025, and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying that this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large dsDNA viruses.IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage, yielding numerous large anucleate viral vesicles that continue to produce virions. Our transcriptome analysis of genome expression in vivo by the Spodoptera frugiperda ascovirus shows that inhibitors of apoptosis are expressed first, enabling viral replication to proceed, after which the SfAV-1a caspase is synthesized, leading to viral vesicle synthesis and subsequent extensive production of progeny virions. Moreover, we detected numerous bicistronic and tricistronic mRNA messages in the ascovirus transcriptome, implying that ascoviruses use other noncanonical translational mechanisms, such as internal ribosome entry sites (IRESs). These results provide the first insights into the molecular biology of a unique coordinated gene expression pattern in which cell architecture is markedly modified, more than in any other known eukaryotic virus, to promote viral reproduction and transmission.