The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

The Complete Genome Sequence of a Second Distinct Betabaculovirus from the True Armyworm, Mythimna unipuncta.

The betabaculovirus originally called Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the characteristic size range and morphology of betabaculovirus granules. Barcoding PCR using cytochrome oxidase I primers with DNA from the MyspGV#8 collection sample confirmed that it had been isolated from the true armyworm, Mythimna unipuncta (Lepidoptera: Noctuidae) and therefore was renamed MyunGV#8. The MyunGV#8 genome was found to be 144,673 bp in size with a nucleotide distribution of 49.9% G+C, which was significantly smaller and more GC-rich than the genome of Pseudaletia unipuncta granulovirus H (PsunGV-H), another M. unipuncta betabaculovirus. A phylogeny based on concatenated baculovirus core gene amino acid sequence alignments placed MyunGV#8 in clade a of genus Betabaculovirus. Kimura-2-parameter nucleotide distances suggested that MyunGV#8 represents a virus species different and distinct from other species of Betabaculovirus. Among the 153 ORFs annotated in the MyunGV#8 genome, four ORFs appeared to have been obtained from or donated to the alphabaculovirus lineage represented by Leucania separata nucleopolyhedrovirus AH1 (LeseNPV-AH1) during co-infection of Mythimna sp. larvae. A set of 33 ORFs was identified that appears only in other clade a betabaculovirus isolates. This clade a-specific set includes an ORF that encodes a polypeptide sequence containing a CIDE_N domain, which is found in caspase-activated DNAse/DNA fragmentation factor (CAD/DFF) proteins. CAD/DFF proteins are involved in digesting DNA during apoptosis.

Genome Sequence of an Alphabaculovirus Isolated from the Oak Looper, Lambdina fiscellaria, Contains a Putative 2-Kilobase-Pair Transposable Element Encoding a Transposase and a FLYWCH Domain-Containing Protein.

The genome sequence of an alphabaculovirus isolated from Lambdina fiscellaria indicated that it is a novel member of a group II lineage. A putative transposable element was identified that contained two genes, including a transposase ortholog. These genes were most closely related to genes of the pea aphid, Acyrthosiphon pisum.

A Distinct Group II Alphabaculovirus Isolated from a Peridroma Species.

The genome sequence of an alphabaculovirus isolated from a Peridroma species indicated that it is a novel member of a group II lineage most closely related to alphabaculoviruses from Spodoptera exigua and Agrotis segetum. It contains a genome of 151,110 nucleotides (nt), with a G+C content of 53.3%.

Functional characterization of hesp018, a baculovirus-encoded serpin gene.

The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects.

Baculovirus nucleocapsid aggregation (MNPV vs SNPV): an evolutionary strategy, or a product of replication conditions?

Lepidopteran nucleopolyhedroviruses are members of the Baculoviridae and have been categorized as having two morphotypes of occluded virions: multiple nucleocapsids or single nucleocapsids within the virion envelope. Although it is a definitive characteristic of specific viruses, it appears to lack a defined genetic basis and is independent of virus phylogeny. This review summarizes the factors that appear to influence this trait and the role that it may play in virus biology.

Genome Sequence of an Alphabaculovirus Isolated from Choristoneura murinana.

The genome sequence of a baculovirus from Choristoneura murinana is 124,689 bp, with a G+C content of 50%, and contains 148 putative open reading frames. The virus is a member of the group I alphabaculoviruses and is most closely related to several other viruses that infect Choristoneura species.

The baculovirus sulfhydryl oxidase Ac92 (P33) interacts with the Spodoptera frugiperda P53 protein and oxidizes it in vitro.

The Autographa californica M nucleopolyhedrovirus (AcMNPV) sulfhydryl oxidase Ac92 is essential for production of infectious virions. Ac92 also interacts with human p53 and enhances human p53-induced apoptosis in insect cells, but it is not known whether any relationship exists between Ac92 and native p53 homologs from insect hosts of AcMNPV. We found that Ac92 interacted with SfP53 from Spodoptera frugiperda in infected cells and oxidized SfP53 in vitro. However, Ac92 did not interact with or oxidize a mutant of SfP53 predicted to lack DNA binding. Silencing Sfp53 expression did not rescue the ability of an ac92-knockout virus to produce infectious virus. Similarly, ac92 expression did not affect SfP53-stimulated caspase activity or the localization of SfP53. Thus, although Ac92 binds to SfP53 during AcMNPV replication and oxidizes SfP53 in vitro, we could not detect any effects of this interaction on AcMNPV replication in cultured cells.

The genome of a baculovirus isolated from Hemileuca sp. encodes a serpin ortholog.

The genome sequence of a baculovirus from Hemileuca sp. was determined. The genome is 140,633 kb, has a G+C content of 38.1 %, and encodes 137 putative open-reading frames over 50 amino acids. 126 of these ORFs showed similarity to other baculovirus genes in the database including all 37 core genes. Of the remaining 11 predicted genes, one is related to a lepidopteran serpin gene. This is the first report of a baculovirus encoding a member of this family of serine protease inhibitors, and to our knowledge the first report of a viral serpin outside the Poxviridae. The genome also contained three homologous repeat sequences. Phylogenetic analysis indicated that the virus is a group II Alphabaculovirus and belongs to a lineage that includes Orgyia leucostigma, Ectropis obliqua, Apocheima cinerarium, and Euproctis pseudoconspersa nucleopolyhedroviruses.

Characterization of baculoviruses from the Martignoni collection.

47 samples from the Martignoni baculovirus collection were characterized by PCR amplification of the lef-8 gene. This led to the identification of sequences from viruses that either were not present in the database, or had been identified, but not further characterized. These included an NPV and a GV from Pseudoletia (Mythimna) unipuncta, and NPVs from Coloradia pandora, the oak and hemlock looper (probably Lambdina sp.), Peridroma sp., the pine butterfly (probably Neophasia sp.), Hemileuca sp., Orgyia vetusta, and several Choristoneura sp. A phylogenetic tree was constructed relating these viruses to their closest relatives in the database.

Analysis of the genome of leporid herpesvirus 4.

The genome of a herpesvirus highly pathogenic to rabbits, leporid herpesvirus 4 (LHV-4), was analyzed using high-throughput DNA sequencing technology and primer walking. The assembled DNA sequences were further verified by restriction endonuclease digestion and Southern blot analyses. The total length of the LHV-4 genome was determined to be about 124 kb. Genes encoded in the LHV-4 genome are most closely related to herpesvirus of the Simplexvirus genus, including human herpesviruses (HHV-1 and HHV-2), monkey herpesviruses including cercopithicine (CeHV-2 and CeHV-16), macacine (McHV-1), bovine herpesvirus 2 (BHV-2), and a lineage of wallaby (macropodid) herpesviruses (MaHV-1 and -2). Similar to other simplexvirus genomes, LHV-4 has a high overall G+C content of 65-70% in the unique regions and 75-77% in the inverted repeat regions. Orthologs of ICP34.5 and US5 were not identified in the LHV-4 genome. This study shows that LHV-4 has the smallest simplexvirus genome characterized to date.

Baculovirus infection induces a DNA damage response that is required for efficient viral replication.

Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10- to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda.

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.

Characterization of Cervidpoxvirus isolates from Oregon, California, and eastern Canada.

The present report describes the analysis of 4 Deerpox virus isolates from California, Oregon, and Ontario, Canada. All 4 isolates were associated with cutaneous crusting lesions. Examination of selected samples by electron microscopy demonstrated that the viruses were morphologically similar to orthopoxviruses. Phylogenetic analysis of the A21 gene, which is found in all poxviruses, indicated that the 4 isolates form a lineage distinct from other members except for those belonging to the genus Cervidpoxvirus of the subfamily Chordopoxvirinae. Members of the Cervidpoxvirus lineage encode a set of genes not found in other poxviruses. These include homologs of genes encoding interleukin 1 receptor antagonists (IL-1Ra) and C-type lectin-like receptors (CTLR). In the current investigation, genes encoding homologs of IL-1Ra and CTLR were amplified from all the isolates and were found to be closely related to orthologs found in the Cervidpoxvirus genus, which further supports the inclusion of these isolates in the Cervidpoxvirus genus.

Characterization of short-lived intermediates produced during replication of baculovirus DNA.

In this report the short-lived DNA replication intermediates produced in both uninfected and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infected Spodoptera frugiperda cells were characterized. The methods used included pulse-labeling of DNA in permiabilized cells, treatment of nascent DNA with Mung bean nuclease, and electrophoresis in neutral and alkaline agarose gels. In contrast to uninfected cells that produced a population of small DNA fragments of about 200bp, a population of heterogeneous fragments of up to 5kb with an average size of 1-2kb derived randomly from the virus genome was identified as the short-lived intermediates produced during AcMNPV replication. The intermediates likely include Okazaki fragments derived from the lagging strands in viral replication forks as well as fragments produced during the recombination-dependent replication.

Diversity of errantivirus (retrovirus) sequences in two cell lines used for baculovirus expression, Spodoptera frugiperda and Trichoplusia ni.

To determine the diversity of retroviruses (errantiviruses) in cell lines used for baculovirus expression, degenerate primers were designed complementary to conserved regions of lepidopteran errantivirus reverse transcriptase genes. These primers were used to PCR amplify sequences from DNA isolated from Spodoptera frugiperda (Sf-9) and Trichoplusia ni (Hi-5) cell lines. Cloning, sequencing, and phylogenetic analysis of over 20 PCR products from each cell line demonstrated the presence of diverse populations of retrovirus sequences comprising at least six major lineages.

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus.

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(6)-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

Baculovirus DNA replication and processing.

In this report, factors involved in baculovirus DNA replication are reviewed. These include factors that are required for DNA synthesis, other factors that have been implicated in genome processing or packaging, and homologs of proteins that are involved in DNA replication or repair in other systems. Conservation of a number of these factors in all baculovirus genomes suggest that many of the observations for specific viral systems may apply to the most if not all members of the Baculoviridae.

Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame.

To investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection-infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production. To better characterize the role of these genes in the baculovirus replication cycle, quantitative DNA replication assays were performed and demonstrated that in cells transfected with the Ac 101, 142, or 144 knockouts, DNA replicated with similar kinetics as a control virus. Western blot analyses of budded virus from cells infected with the repair viruses showed that these proteins are associated with the viral nucleocapsid. Furthermore, immunoelectron microscopy of cells transfected with the knockout bacmids revealed defects in nucleocapsid production for all three constructs. From these results we concluded that the gene products encoded from these open reading frames are essential for virus production and may be involved in DNA processing, packaging, or nucleocapsid morphogenesis.

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.