Sequence and comparative analysis of the genome of HSV-1 strain McKrae.

Ocular infection by HSV-1 strain McKrae is neurovirulent in both mice and rabbits and causes fatal encephalitis in approximately 50% of animals. In addition, it spontaneously reactivates with high frequency relative to other HSV-1 strains in rabbits. We sequenced the McKrae strain genome and compared its coding protein sequences with those of six other HSV-1 strains. Most of the 74 predicted protein sequences are conserved; only eleven are less than 98% conserved. Eight proteins were identified to be unique for McKrae based on sequence homology bit score ratio (BSR). These include five proteins showing significant variations (RL1, RS1, UL49A, US7 and US11), two truncated proteins (UL36 and UL56) and one (US10) containing an extended open reading frame. The McKrae strain also has unique features in its 'a' sequence and non-coding sequences, such as LAT and miRNA. These data are indicative of strain variation but need further work to connect observed differences with phenotype effects.

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

Characterization of a novel alphaherpesvirus associated with fatal infections of domestic rabbits.

A virus was found to be associated with a severe disease affecting rabbits on a farm near Anchorage, Alaska. Extracts from the skin of infected rabbits produced syncytia and cell lysis in cultured rabbit skin, rabbit kidney, and Vero cells. Examination of the infectious agent by electron microscopy revealed an icosahedral nucleocapsid surrounded by an envelope with a diameter of about 120 nm, suggesting that it was a herpesvirus. The viral genome was determined to be composed of double-stranded DNA of 120-130 kbp. PCR using degenerate primers to conserved herpesvirus genes was used to amplify sequences from purified viral DNA. Sequencing of these products allowed the design of specific primers so that complete sequence data for a number of genes could be determined. Analysis of these data indicated that the virus is most closely related to bovine herpesvirus 2. The next most closely related viruses are human herpesviruses 1 and 2, and a number of cercopithecine herpesviruses. Experimental exposure of domestic rabbits to the isolate resulted in severe clinical disease and necrosis in the spleen and lymph node. In addition, viral DNA was identified in a variety of tissues by PCR, consistent with a systemic infection. Taken together, these data suggest that this virus is highly pathogenic for domestic rabbits and belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus.

Analysis of the genome sequence of an alpaca coronavirus.

Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3' end. This virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs) characteristic of Group 2 bovine coronavirus (BCoV). Phylogenetic analysis showed that the ACoV genome is clustered closely (>99.5% identity) with two BCoV strains, ENT and LUN, and was also closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (>96%), and porcine hemagglutinating encephalomyelitis virus (>93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT, respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same ancestor as BCoV ENT and LUN.

On the classification and nomenclature of baculoviruses: a proposal for revision.

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Characterization of a baculovirus with a deletion of vlf-1.

Very late expression factor (VLF-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for high levels of expression of the very late genes p10 and polh, and evidence suggests VLF-1 may also be involved in viral DNA replication. In this study, investigations determined whether VLF-1 is essential for viral DNA replication by generating a vlf-1 knockout bacmid containing the AcMNPV genome through homologous recombination in Escherichia coli. Additionally, a vlf-1 repair bacmid was constructed by transposing the vlf-1 ORF and native promoter region into the polh locus of the vlf-1 knockout bacmid. After transfecting these virus constructs into Spodoptera frugiperda (Sf-9) cells, the vlf-1 knockout bacmid was unable to produce a viral infection while the repair bacmid propagated at wild-type levels. Experiments were performed to conclude whether the vlf-1 knockout phenotype was due to a defect in viral DNA synthesis or late gene transcription. Southern blot analyses determined that the vlf-1 knockout bacmid was able to replicate viral DNA but only to about one-third the level of wild-type or rescued controls. In addition, virion DNA was not detected in the supernatant of transfected cells, indicating that the DNA synthesized by the mutant virus was unable to assemble into virions that bud out of the cells. Analysis of viral gene transcription confirmed that late gene transcription was not affected by the vlf-1 knockout but transcription of the very late gene p10 was substantially reduced.

Relatedness of baculovirus and gypsy retrotransposon envelope proteins.

BACKGROUND: Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins. One group utilizes gp64, a low pH-dependent envelope fusion protein, whereas the other employs a protein family (e.g. LD130 in the Lymantria dispar nucleopolyhedrovirus) unrelated to gp64, but that is also low pH-dependent. Database searches with members of the LD130 protein family often record significant levels of homology to envelope proteins from a number of insect retrovirus-like transposable elements of the gypsy class. In this report, the significance of the homology between these two types of envelope proteins is analyzed. RESULTS: The significance of the alignment scores was evaluated using Z-scores that were calculated by comparing the observed alignment score to the distribution of scores obtained for alignments after one of the sequences was subjected to 100 random shuffles of its sequence. These analyses resulted in Z-scores of >9 for members of the LD130 family when compared to most gypsy envelope proteins. Furthermore, in addition to significant levels of sequence homology and the presence of predicted signal sequences and transmembrane domains, members of this family contain a possible a furin cleavage motif, a conserved motif downstream of this site, predicted coiled-coil domains, and a pattern of conserved cysteine residues. CONCLUSIONS: These analyses provide a link between envelope proteins from a group of insect retrovirus-like elements and a baculovirus protein family that includes low-pH-dependent envelope fusion proteins. The ability of gypsy retroelements to transpose from insect into baculovirus genomes suggests a pathway for the exchange of this protein between these viral families.

Characterization of a baculovirus-encoded protein that is associated with infected-cell membranes and budded virions.

Two types of envelope fusion proteins have been identified in lepidopteran baculoviruses. GP64 is found in Autographa californica multinucleocapsid nucleopolyhedrovirus, Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (OpMNPV), and other relatively closely related viruses, while Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), which lacks GP64, utilizes LD130 as its envelope fusion protein. Homologs of ld130 have since been found not only in all the sequenced gp64-minus virus genomes, but also in the genomes of gp64-containing viruses. In addition, they are evolutionarily related to the envelope proteins of certain insect retroviruses. In this report, the characterization of a LD130 homolog (OP21) from OpMNPV, which also contains gp64, is described. Western blot analysis of extracts of OpMNPV-infected Lymantria dispar cells, using antibodies generated against OP21, identified an infected cell-specific doublet of 85 and 89 kDa. These bands were first observed at about 6 h p.i. and were present at all later time points. Such analyses also demonstrated that OP21 was associated with budded virions. Tunicamycin treatment of OpMNPV-infected cells indicated that OP21 is N-glycosylated. Studies employing NP-40 to remove the envelope from budded virions indicated that the majority of OP21 remained associated with the nucleocapsid fraction, whereas all GP64 was removed. Confocal immunofluorescence microscopy showed that OP21 and GP64 have a similar pattern of distribution on the membrane of cells infected with OpMNPV. Immunoelectron microscopy of budded virions also showed similar patterns of localization for both OP21 and GP64.

The Autographa californica nucleopolyhedrovirus IE-1 protein complex has two modes of specific DNA binding.

Missing contact footprinting with formic acid as a modifying reagent was used to examine specific IE-1 binding contacts to double-stranded oligonucleotides that contained either a consensus hr repeat sequence or a sequence from the pe38 promoter, which is down regulated by IE-1. The hr repeat sequences contain two consensus IE-1 binding motifs (IBMs) flanking a central EcoRI site that are oriented in opposite directions with respect to each other. IE-1 was found to contact regions including both IBMs. The bases footprinted in the top strand included the left IBM (IBM-A), whereas bases in the bottom strand were footprinted in a region that included IBM-B and part of IBM-A. When substitution mutations were introduced into either IBM, bases on both strands of the remaining IBM were strongly footprinted. As with the hr IBM-mutant constructs, bases footprinted in the pe38 promoter construct included both strands of the single IBM.

Identification of the lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae.

The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.

Characterization of a baculovirus alkaline nuclease.

All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in the Herpesviridae. In this report we describe the characterization of the alkaline nuclease (AN) homolog of the Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg(2+) over Mn(2+), had optimal activity at 35 degrees C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.

Identification of additional genes that influence baculovirus late gene expression.

We were unable to confirm transient late gene expression using constructs of 18 genes that had been reported to support Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) late gene expression when transfected into Spodoptera frugiperda cells [Lu, A., and Miller, L. K. (1995). J. Virol. 69, 975-982]. Three genes (orf66, orf68, and orf41) were included, all or in part, in the constructs used in that study, but they had not been independently tested. Therefore we investigated these and neighboring orfs for their influence on late gene expression. We found that orf41 was required for late gene expression and that sequences within orf45 appeared to be required for the expression of orf41. Although orf66 and orf68 did not appear to affect late gene expression, orf69 stimulated expression. orf69 was found to have high homology to recent entries in GenBank from a variety of organisms. In addition, it was found that orf121, which was shown to be involved in early gene expression, and the viral homolog of pcna did not influence late gene expression.

Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143).

LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar.

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

Characterization of a baculovirus-encoded ATP-dependent DNA ligase.

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.

AcMNPV late expression factor-5 interacts with itself and contains a zinc ribbon domain that is required for maximal late transcription activity and is homologous to elongation factor TFIIS.

The late expression factor-5 gene (lef-5) of Autographa californica multinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione-S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a beta-glucuronidase (GUS) reporter gene under control of the late vp39 capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+ caused a reduction in activity similar to that when the domain was eliminated completely.

Differential stability of baculovirus late transcription complexes during initiation and elongation.

An in vitro system for baculovirus late transcription was developed that allows comparison of conditions that affect transcription initiation and elongation. A series of synthetic promoters was constructed based on the baculovirus late p6.9 promoter. The modified promoters were designed with a cytidine-free region downstream of the late promoter in order to yield paused transcripts of defined lengths in the absence of CTP. Transcription was found to be more efficient from a supercoiled template than from a linear template for this promoter. The stalled transcription complex remained competent and could be elongated in the presence of a full set of nucleotides. This made it possible to separately test the effects of heat treatment and inhibition by sarkosyl and heparin on initiation and elongation. Elongation complexes were more resistant than initiation complexes to each of these treatments. Furthermore a 1-2 mM MgCl2 concentration is critical for optimal initiation, but elongation can proceed in the presence of MgCl2 concentrations as high as 20 mM.

Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of the L dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer did not show increased activity when cotransfected with LdMNPV ie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system. LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line.

A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with BglII sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (pIe-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1-specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.