D. Zuidema

Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64

ABSTRACT GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector.

An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.

DISTINCT GENE ARRANGEMENT IN THE BUZURA SUPPRESSARIA SINGLE-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS GENOME

The genome organization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was largely elucidated and compared to those of other baculoviruses. A detailed physical map was constructed for the restriction enzymes BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, XbaI and XhoI. The 120.9 kbp viral genome was cloned as restriction fragments into a plasmid library from which about 43.5 kbp of dispersed sequence information was generated. Fifty-two putative open reading frames homologous to those of other baculoviruses were identified and their location in the genome of BusuNPV was determined. Although the gene content of BusuNPV is similar to that of Autographa californica multiple-nucleocapsid nucleopolyhedrovirus, Bombyx mori nucleopolyhedrovirus and Orgyia pseudotsugata multiple-nucleocapsid nucleopolyhedrovirus, the gene order is, however, significantly different from that observed in the other viruses, which have a high degree of collinearity. A new approach (GeneParityPlot) was developed to represent the differences in gene order among baculoviruses when limited sequence information is available and to take advantage of the high degree of gene conservation. The data obtained show that BusuNPV is a distinct baculovirus species and the analyses suggest that gene distribution along baculovirus genomes may be used as a phylogenetic marker.

Localization of the 34-kDa polyhedral envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus.

SummaryUsing immuno-electron microscopy the 34 kDa polyhedron envelope (PE) phosphoprotein (pp 34) was localized in cells infected withAutographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV). In wild-type AcMNPV-infected cells this protein was found associated with electron-dense “spacers” and the polyhedron envelopes demonstrating their structural relationship. In these cells pp 34 was also found associated with fibrillar structures present in the nucleus and cytoplasm of infected cells. However, when cells were infected with an AcMNPV mutant with an inactivated pp 34 gene, antiserum against pp 34 still localized in fibrillar structures. In cells infected with AcMNPV mutants lacking p 10, and thus devoid of fibrillar structures, pp 34 localized normally with both electron-dense “spacers” and polyhedron envelopes. These data confirm that fibrillar structures are not essential for the morphogenesis of polyhedron envelopes and demonstrate that the association of anti pp 34 serum with these structures is specific but fortuitous.

Isolation of a Spodoptera exigua baculovirus recombinant with a 10.6 kbp genome deletion that retains biological activity.

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13.7 and 21.6 map units of SeMNPV and including ecdysteroid UDP glucosyl transferase, gp37, chitinase and cathepsin genes, as well as several genes unique to SeMNPV. The result indicated, however, that these genes are dispensable for virus replication both in vitro and in vivo. A mutant with a similar deletion was identified by PCR in the parental wild-type SeMNPV isolate, suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses. The generation of recombinants in vivo, combined with the alternate cloning between insects and insect cells, is likely to be applicable to many baculovirus species in order to obtain biologically active recombinants.

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.

Identification and functional analysis of a non-hr origin of DNA replication in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus.

The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) was screened for the presence of putative origins of DNA replication (oris). Using a transient DNA replication assay, several fragments were identified that underwent SeMNPV-dependent DNA replication in Spodoptera frugiperda cells (Sf-AE-21). Preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). Restriction fragment Xbal-F2 showed a distinct sequence reminiscent of Autographa californica and Orgyla pseudotsugata MNPV (AcMNPV and OpMNPV) non-hr oris. Deletion analysis of this fragment indicated that the essential sequences of this putative non-hr ori mapped within a region of 800 bp. Sequence analysis of this region showed a unique distribution of six different (im)-perfect palindromes, several polyadenylation motifs and the occurrence of multiple direct repeats. No sequence homology or similarities to other reported baculovirus oris were detected. The spatial and modular distribution of these motifs are similar to those of the non-hr oris of AcMNPV and OpMNPV. Comparison of baculovirus non-hr and consensus eukaryotic oris revealed no consensus ori but indicated that each of the non-hrs studied so far is unique. From the structural similarity, however, it was concluded that the SeMNPV Xbal-F2 ori represents a baculovirus non-hr type ori. In addition, evidence is provided that SeMNPV renders more specificity to baculovirus DNA replication than AcMNPV.

High frequency recombination between homologous baculoviruses in cell culture

Summary. The frequency of recombination between homologous baculoviruses was investigated in cell culture upon coinfection with two Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants. These parental recombinants differed at two loci, separated by 20 kb, each carrying a different marker. The progeny recombinants generated by homologous recombination were easily distinguishable by the presence or absence of these markers. The mean percentage of the newly generated recombinants relative to a single locus varied between 23 and 41%, depending on the multiplicity of infection used for coinfection. These results provide further evidence that homologous recombination occurring during baculovirus replication is a highly frequent event.

Specificity of multiple homologous genomic regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.

The region upstream of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ubiquitin gene contains four near-identical 68-bp-long palindromic repeats. This region, named Sehr6 and located at map unit (m.u.) 88 of the SeMNPV genome on pSeEcoRI-2.2, showed structural homology to previously identified homologous regions (hrs) in a number of other baculoviruses. Hrs function as enhancers of transcription and as putative origins (oris) of baculovirus DNA replication. Five additional hrs (Sehr1-Sehr5) were identified on the SeMNPV genome by Southern blot hybridization with an 18-bp-long oligonucleotide complementary to a sequence conserved within the arms of the four palindromic repeats of Sehr6. Sehr1-Sehr6 were dispersed on the SeMNPV genome at m.u. 8.0, 30.0, 38.5, 51.0, 77.0 and 88.0, respectively. Sequence analysis of these hrs confirmed the presence of palindromic repeats, highly similar to those found in pSeEcoRI-2.2. The number of palindromes varied from one (Sehr4) to nine (Sehr1) per hr. The Sehrs are all present in non-coding regions of the SeMNPV genome and also contain multiple putative transcription recognition sequences. Plasmids containing either of the Sehrs replicated in an SeMNPV-dependent DNA replication assay. The Sehrs were unable to replicate in an AcMNPV-dependent DNA replication assay. This was in contrast to the previously observed SeMNPV non-hr type ori, which replicated in the presence of both AcMNPV and SeMNPV. These data suggest that the replication of SeMNPV and the role of hrs in this process is highly specific.

Specificity of polyhedrin in the generation of baculovirus occlusion bodies

The role of polyhedrin in the occlusion of virions was studied by substituting two heterologous polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.