Characterization of Actinidia virus 1, a new member of the family Closteroviridae encoding a thaumatin-like protein.

A new member of the family Closteroviridae was detected in Actinidia chinensis grown in Italy, using next generation sequencing of double-stranded RNA. The virus isolate, named Actinidia virus 1 (AcV-1) has a genome of 18,848 nts in length, a structure similar to the unclassified persimmon virus B (PeVB) and contains 12 open reading frames (ORFs) greater than 6 KDa, one carrying two papain-like leader proteases, a methyltransferase, a helicase and an RNA-dependent RNA polymerase domain. Additional ORFs code for homologs of heat shock protein 70, heat shock protein 90 and a coat protein. Curiously, AcV-1 and PeVB genomes code for a thaumatin-like protein, a peculiarity unreported for other viruses. In phylogenetic analyses both viruses group in a distinct clade evolutionarily related to closteroviruses. The final taxonomic position of AcV-1 within the family Closteroviridae is yet to be clarified.

Comparison of complete nucleotide sequences and genome organization of six distinct cherry leaf roll virus isolates from New Zealand.

The complete genomic sequences of six phenotypically distinct cherry leaf roll virus (CLRV) isolates from New Zealand were determined and compared. RNA1 and RNA2 are 7,919-7,921 and 6,361-6,363 nucleotides in length, respectively, excluding the poly(A) tails. Both genome segments contain a single open reading frame and encode one polyprotein of 2,113 amino acids for RNA1 and 1,590 aa for RNA2, with corresponding molecular masses of 235.5-236.1 kDa and 174.2-174.8 kDa, respectively. The six RNA1 sequences share 92.0-99.7 % nt and 97.0-99.5 % aa similarity, and the RNA2 sequences share 91.4-99.8 % nt and 94.3-99.7 % aa similarity. Phylogenetic analysis established close associations between the six New Zealand CLRV isolates and members of the subgroup C nepoviruses.

Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana.

Real-time quantitative polymerase chain reaction (qPCR) of complementary DNA is now a standard method for studies of gene expression. However, qPCR can identify genuine variation only when transcript quantities are accurately normalized to an appropriate reference. To identify the most reliable reference genes for transcript quantification by qPCR, we describe a systematic evaluation of candidate reference genes of Arabidopsis thaliana ecotype Columbia-0 (Col-0). Twelve genes were selected for transcript stability studies by qPCR of complementary DNA prepared from Arabidopsis leaf tissue infected with one of five plant viruses (Cauliflower mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, Turnip mosaic virus, and Turnip yellow mosaic virus). The F-box family protein, elongation factor 1-α, sand family protein, and protodermal factor 2 gene transcripts showed the most stable accumulation, whereas a traditionally used reference gene, Actin8, showed the least stable accumulation as measured by the geNorm algorithm. The data furnish plant virologists with reference genes for normalization of qPCR-derived gene expression in virus-infected Arabidopsis and will be beneficial to the selection and design of primers targeting orthologous genes in other plant species.

Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain.

The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host-virus interactions, and the mechanism of resistance in P. trifoliata.

Complete genome sequences of two distinct and diverse Citrus tristeza virus isolates from New Zealand.

Two Citrus tristeza virus (CTV) isolates from New Zealand that display distinct phenotypes were isolated, examined and sequenced in full. The first isolate, NZ-M16, is largely asymptomatic and non-transmissible by the aphid vector Toxoptera citricida, while the second, NZ-B18, is highly transmissible and induces very severe symptoms on C. sinensis and C. aurantii. Phylogenetic analysis of the genome sequences showed that both isolates were approximately 90-93% similar to the VT and T318 isolates but possessed only 89% identity to one another. Based on sequence identity, both isolates are VT subtypes, with NZ-M16 being T3-like, while NZ-B18 is a member of a novel subtype with B165 from India.

First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand.

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5'-GCACGGCAGATAAACACGAC-3') and reverse (5'-GTGGGCAACCTTCAACTGTG-3') primers designed to amplify the 3' untranslated region (3'UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3'UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

First Report of Citrus leaf blotch virus in New Zealand.

Despite a high incidence of Citrus tristeza virus (CTV) in citrus in New Zealand, viral diseases have had only a minor impact on the New Zealand citrus industry, largely because of the use of Poncirus trifoliata and hybrid rootstocks derived from this. In August of 2007, a PCR-based survey for seven citrus viruses was conducted on 104 commercial orchard trees that represented a range of Citrus scion species, as well as P. trifoliata and P. trifoliata × Citrus sinensis rootstock, grown from imported and local budwood or from seed in the case of rootstocks, from the citrus-growing regions of Kerikeri, Tauranga, and Gisborne. Total RNA was extracted from young, green bark and leaf tissue from each source. Using a primer pair amplifying a 425-bp region of the Citrus leaf blotch virus (CLBV) coat protein gene (sense: 5'-AGCCATAGTTGAACCATTCCTC-3' and antisense: 5'-GCAGATCATTCACCACATGC-3'), 26 (25%) of the plant samples yielded a DNA fragment of the size expected for CLBV, including 6 of 21 C. sinensis, 1 of 2 C. limon, 3 of 10 C. unshiu, and 5 of 7 C. paradisi scion samples and 5 of 9 P. trifoliata and 6 of 9 P. trifoliata × C. sinensis rootstock samples. Identification of CLBV (an unclassified member of the family Flexiviridae) was confirmed by amplification of a second region of the genome of 1,045 bp spanning the ORF2/ORF3 domains (sense: 5'-ATGAAAAGCCAGTTATGCACCA-3' and antisense: 5'-CTCAGCATTCCCAGGAATAACC-3'). A subset of the PCR products for the CP and ORF2/ORF3 fragments were sequenced for analysis (GenBank Accession nos. EU670243-EU670251 and EU862284-EU862288). Sequences from all CLBV isolates showed 97 to 99% nucleotide identity with the CLBV reference isolate SRA-153 (GenBank Accession No. AF318061) (1) in the coat protein fragment and 98 to 99% nucleotide identity for the partial ORF2/ORF3 fragment. The bud-union crease symptom reported to be caused by CLBV was observed only on one C. paradisi scion on P. trifoliata rootstock, while the remaining samples were either asymptomatic or symptoms were attributed to co-infection with CTV. Since CLBV-infected plants were found from all major growing regions, it is apparent that CLBV is widespread in New Zealand, although it is not known where in this country it may have originated. Reference: (1) M. C. Vives et al. Virology 287:225, 2000.

Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect vanilla.

Sequence was determined for the coat protein (CP) gene and 3' non-translated region (3'NTR) of two vanilla mosaic virus (VanMV) isolates from Vanilla tahitensis, respectively from the Cook Islands (VanMV-CI) and French Polynesia (VanMV-FP). Both viruses displayed distinctive features in the N-terminal region of their CPs; for VanMV-CI, a 16-amino-acid deletion including the aphid transmission-related DAG motif, and for VanMV-FP, a stretch of GTN repeats that putatively belongs to the class of natively unfolded proteins. VanMV-FP CP also has a novel DVG motif in place of the DAG motif, and an uncommon Q//V protease cleavage site. The sequences were compared to a range of Dasheen mosaic virus (DsMV) strains and to potyviruses infecting orchids. Identity was low to DsMV strains across the entire CP coding region and across the 3'NTR, but high across the CP core and the CI-6K2-NIa region. In accordance with current ICTV criteria for species demarcation within the family Potyviridae, VanMV-CI and VanMV-FP are strains of DsMV that exclusively infect vanilla.

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant 'potex-like' viruses.

This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3' terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant 'potex-like' viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant 'potex-like' coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant 'potex-like' virus group, distinct from BVF.

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.

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.

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.

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.

Identification and characterization of a second putative origin of DNA replication in a baculovirus of Orgyia pseudotsugata.

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.

Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.

Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.

Detection of an unidentified potyvirus from Roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant DNA sequences.

Degenerate potyvirus-specific primers were used in the PCR to amplify cDNA representing a 335 nucleotide region of the coat protein gene in RNA purified from an uncharacterised potyvirus isolated from Roystonea regia palms in Queensland, Australia. The RNA was also detected by PCR in total nucleic acid extracts from infected Nicotiana benthamiana, N. clevelandii and Vicia faba. The method also successfully detected pea seed-borne mosaic virus in Pisum sativum. In addition the procedure amplified DNA's of approximately 200 bp and 420 bp, of non-viral origin, from total nucleic acid extracts of healthy Nicotiana spp, indicating that size of the PCR products needs to be included as a criterion for identifying virus specific products when this method is used.

Identification and characterization of a putative origin of DNA replication in the genome of a baculovirus pathogenic for Orgyia pseudotsugata.

A four-kilobase (kb) region (HindIII-N, map units 7.0-11.3) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. The plasmid DNA appeared to be replicated to form high molecular weight multimers. Plasmids with deletions of up to 1.8 kb from either end of the HindIII-N region were replication competent. However, a discrete sequence, contained within the region bracketed by the deletions, capable of specifying replication was not identified. No evidence for sequence homology was found between the OpMNPV HindIII-N region and regions elsewhere in the OpMNPV genome or to putative Autographa californica MNPV (AcMNPV) replication origins. Origin-dependent plasmid replication was shown to require the presence of the OpMNPV DNA polymerase gene. The OpMNPV origin replicated poorly in AcMNPV-infected Spodoptera frugiperda cells and, conversely, a putative AcMNPV origin (hr2) replicated at low levels in OpMNPV-infected Lymantria dispar cells.

A baculovirus encoded 16-kDa glycoprotein localizes near the nuclear membrane of infected cells.

An open reading frame (ORF 2) located upstream of the polyhedron envelope protein gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) was cloned in frame into a trpE expression vector. The fusion protein produced by this construct was used for the production of a monospecific antiserum. Western blot analysis of OpMNPV-infected Lymantria dispar cells detected a 16-kDa protein at 24 hr postinfection. The 16-kDa protein was determined to be N-glycosylated by tunicamycin treatment of infected cells. Immunofluorescence microscopy localized the 16-kDa protein to foci of intense cytoplasmic staining near the nuclear membrane. Immunoelectron microscopy indicated that the 16-kDa protein is associated with lamellar-like structures peripheral to the nuclear membrane and with envelopes of virus that have budded into the cytoplasm. The 16-kDa protein was not associated with extracellular budded or polyhedron-derived virions.