In vitro host range of Autographa californica nucleopolyhedrovirus recombinants lacking functional p35, iap1 or iap2.

We have examined the host range in different insect cell lines of Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants lacking p35, iap1 or iap2. These genes encode, or are predicted to encode, anti-apoptotic proteins. Abrogation of p35 reduced the ability of AcMNPV to replicate in permissive cell lines derived from Spodoptera frugiperda insects by inducing apoptosis. In semi-permissive cell lines, such as Lymantria dispar and Spodoptera littoralis cells, we observed cytopathic effects after infection with AcMNPV but little virus production. Infection of these cells by AcMNPV lacking p35 resulted in apoptosis. However, p35-deficient viruses were still able to replicate normally in Trichoplusia ni, Mamestra brassicae and Panolis flammea cell lines. Disruption of AcMNPV iap1 and iap2 was found not to affect virus replication in any of the cell lines. It was also possible to disrupt both iap1 and iap2 in the same virus without loss of infectivity. A virus without iap1 and p35 demonstrated identical growth characteristics and host range to a virus lacking p35. We conclude that in cells which respond to AcMNPV infection by initiating programmed cell death, the p35 gene product alone is sufficient to inhibit apoptosis. Removal of iap1 or iap2 has no effect on virus replication, even in cell lines which do not undergo apoptosis in response to AcMNPV infection. Our results with two semi-permissive cell lines further indicate that whilst p35 is important in blocking block apoptosis, other factors are involved in restricting AcMNPV replication within these cells.

The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.

A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.

The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.

The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence data confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.

Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region.

The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the C6 strain of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. These data provide a complete description of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into the AcMNPV. Ten potential open reading frames (ORFs) were identified in the complete sequence, on either strand of DNA. The largest of these was 1629 nucleotides in length and was located downstream from the polyhedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified an RNA of 2000 nucleotides which was produced in infected cells from 12 hr p.i. and remained until at least 48 hr p.i. S1 nuclease mapping and analysis of cDNA clones located the 3' end of the mRNA at a site 16 nucleotides downstream of the polyhedrin coding sequences. The 5' end of the mRNA was mapped using primer extension analysis of polyadenylated RNA. The mRNA start site was positioned within a late/very late consensus transcription initiation motif (ATAAG), 428 nucleotides upstream from the potential ATG translation initiation codon. The biological significance of the putative gene product was assessed by inserting a synthetic oligonucleotide in the carboxyl terminal coding sequences of ORF 8 (1629) to prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication.

Genomic sequence of the structural proteins of louping ill virus: comparative analysis with tick-borne encephalitis virus.

The genomic RNA of louping ill virus coding for capsid, premembrane, membrane, and envelope proteins was cloned and sequenced. Hydrophilicity profiles of the deduced amino acid sequence shared homologous functional domains with other flaviviruses. The premembrane and envelope proteins contain N-glycosylation sites and conserved cysteine residues which are important for maintaining the secondary structures of the proteins. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. With the capsid and membrane proteins, the degree of homology between louping ill and the western subtype was greater than that between the two subtypes, indicating very close evolutionary relationships between louping ill and the western subtype of tick-borne encephalitis. Thus, louping ill and tick-borne encephalitis may be varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and our sequence data on louping ill virus, we have identified three discontinuous peptides (amino acids 81-88, 207-212, and 230-234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses and may provide a specific genetic marker for these viruses.

Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.

Isolation of a bisegmented double-stranded RNA virus from Thirlmere reservoir.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

Induction of a nonoccluded baculovirus persistently infecting Heliothis zea cells by Heliothis armigera and Trichoplusia ni nuclear polyhedrosis viruses.

A nonoccluded singly enveloped baculovirus (baculovirus X) persistently infects Heliothis zea (IMC-HZ-1) cells in culture. Singly enveloped nuclear polyhedrosis viruses from H. zea and Heliothis armigera, and multiply enveloped nuclear polyhedrosis viruses from Trichoplusia ni, Spodoptera frugiperda, and Spodoptera littoralis were all found to induce baculovirus X. Experiments are reported which use metabolic inhibitors and inactivated inducing virus to show that it is probable that a structural component of the virus, most likely a protein, is responsible for inducing baculovirus X. The persistent virus is induced to replicate by uv-inactivated virus but not by heat-inactivated inducing virus. The virus is not induced to replicate by a number of metabolic inhibitors in the absence of an inducing virus. Inhibition of transcription and translation prevents the induction of the persistent virus by an inducing virus. Inhibition of DNA replication has no effect on the induction of the virus. This suggests that the persistent virus genome is present in abundance in all cells.