Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.     

The Autographa californica nuclear polyhedrosis virus p143 gene encodes a DNA helicase

The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a K(m) of 60 microM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.     

Quantitative analysis of cytoplasmic actin gene promoter and nuclear polyhedrosis virus immediate-early promoter activities in various tissues of silkworm Bombyx mori using recombinant Autographa californica nuclear polyhedrosis virus as vector

Cassettes harboring luciferase reporter driven by Bombyx mori cytoplasmic actin gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcΔEGT to generate the recombinant Autographa californica nuclear polyhedrosis viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses were injected into the hemocoele of newly ecdysed 5th instar larvae. The activities of the A3 and IE-1 promoters in various tissues were measured by luciferase activity assay and normalized by the copy number of recombinant virus. Results showed that the activity of the A3 promoter was approximately 10-fold higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were highest in the silk gland, followed by fat body, middle gut, Malpighian tubule, and hemocyte. In silk gland, activity of the two promoters was highest in posterior silk gland, followed by middle and anterior silk glands. The difference in promoter activities reflects the growth speed of tissue in silkworm larvae. The activity of the A3 promoter remained unchanged and was not inhibited significantly by viral factors at least 3–4 d post injection of rAcNPV.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.     

Concentrating Autographa californica nuclear polyhedrosis virus and recombinant alkaline phosphatase from insect cells using a temperature-sensitive hydrogel

Recombinant alkaline phosphatase expressed in insect cells was concentrated by a factor of one and half times at a separation efficiency of 54.2% using hydrogel ultrafiltration. Enzyme concentration was confirmed by SDS-PAGE as well as by spectrophotometric measurement. Wild and recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) were concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein.

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. As a first step in the functional characterization of viral replication proteins, we purified a single-stranded DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was eluted from the columns at 0.9 to 1.0 M NaCl. This protein was not evident in extracts prepared from control cells, suggesting that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-stranded vector DNA competed as efficiently as single-stranded DNA containing the AcNPV origin of DNA replication. Competition binding experiments indicated that SSB showed a preference for single-stranded DNA over double-stranded DNA. To determine whether SSB was encoded by the lef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indicated that LEF-3 produced in bacteria or in rabbit reticulocyte lysates specifically reacted with antiserum produced by immunization with purified SSB. Immunoblot analyses of infected cell extracts revealed that SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.     

Replication ofAutographa californica nuclear polyhedrosis virus in a thymidine kinse deficientSpodoptera exigua cell line (SE-UCR-1A)

Summary Cultivation of aSpodoptera exigua cloned cell line (SE-UCR-1A) for 8 to 9 mo. in a medium containing increasing amounts of bromodeoxyuridine (BUdr) resulted in the selection of a BUdr-resistant subline unable to grow in TNMFH medium supplemented with HAT (hypoxanthine, 10⁻⁴ M; aminopterin, 10⁻⁷ M; thymidine, 10⁻³ M). Subsequent assay of this subline revealed an absence of thymidine kinase (TK) activity. The specific activity of the wild-type (wt) cells was 878±192 counts per min (cpm)/μg supernatant protein compared to 9 cpm/μg for the BUdr-resistant, HAT-sensitive subline. In addition the wt activity was inhibited >90% by addition of BUdr to the assay, indicating that the activity is predominantly due to TK and not to a nonspecific nucleoside phosphotransferase. The morphology of the TK-deficient (−) cells was indistinguishable from that of wt cells. The doubling time for wt cells in TNMFH was 16 h; however, in TNMFH-HAT it was 36 h. In comparison the TK(−) cells in TNMFH had a doubling time of 61 h. Cultivation of TK(−) cells in nonselective TNMFH for 14 mo. to date has not changed the TK(−) characteristic of the subline. The host-cell TK was not required for development of progeny virus fromAutographa californica NPV inoculum. Although similar numbers of cells were infected (80 to 90%) in the wt and TK(−) lines and extracellular virus was generated at similar rates to similar titers in both media, the initial appearance of virus in the medium of TK(−) cell was delayed 10 to 20 h compared to wt cells. In addition, polyhedra appearance was similarly delayed in TK(−) cells and only 20 to 25% of the cells contained >10 polyhedra per cell compared to 75 to 90% for wt cells. Also, during infection of wt cells, specific activity of TK increased twofold peaking at 20 to 30 h postinfection, yet there was no stimulation of TK activity in infected TK(−) cells.     

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).     

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.     

The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.