Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N′-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33°C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33°C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33°C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.     

Construction of a Genetic Map of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus by Marker Rescue of Temperature-Sensitive Mutants

Mutations of seven temperature-sensitive mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (NPV) were mapped with respect to the physical restriction map of the A. californica NPV DNA by marker rescue. DNAs from two distantly related NPVs of the multiply embedded type and two NPVs of the singly embedded type were unable to rescue two A. californica NPV mutants.     

Protein Kinase Activity Associated with the Extracellular and Occluded Forms of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Protein kinase activity is associated with both the extracellular and the occluded forms of Autographa californica nuclear polyhedrosis virus, a baculovirus. Serine and threonine are the predominant amino acids phosphorylated by the kinase activity associated with both viral forms; no phosphotyrosine was detected. The addition of calcium, cAMP, or cGMP has no apparent effect on the amount of phosphorylation or the substrates phosphorylated.     

Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus

A physical map of the 88 × 10⁶ dalton, circular DNA genome of Autographa californica nuclear polyhedrosis virus was constructed. The complete order of BamHI and XmaI restriction enzyme sites was determined. The EcoRI and HindIII fragments were partially ordered, and their general locations, relative to the BamHI and XmaI maps, were determined. Alterations in the restriction endonuclease fragment patterns of natural genotypic variants of A. californica nuclear polyhedrosis virus, including Trichoplusia ni MEV nuclear polyhedrosis virus, were located on the physical map. Alterations were found throughout the A. californica nuclear polyhedrosis virus DNA genome.     

Generalized Immunoassay for Autographa californica Nuclear Polyhedrosis Virus Infectivity In Vitro

A quantitative in vitro immunoassay for the infectivity of Autographa californica nuclear polyhedrosis virus was developed and performed in six different lepidopteran cell lines. The assay was not dependent upon cytopathic effect or polyhedron production, but rather upon viral antigen production and its recognition in a peroxidase-antiperoxidase staining procedure. The importance of using such an assay for accurately assessing infectivity in cell lines which produce polyhedra inefficiently was demonstrated. Differences among the cell lines in sensitivity to viral infection were clearly shown. Differences in the time required to produce infectious progeny were also noted among cells of the same cell line.     

Physical Maps of Autographa californica and Rachiplusia ou Nuclear Polyhedrosis Virus Recombinants

TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.     

Orientation of the Genome of Autographa californica Nuclear Polyhedrosis Virus: a Proposal

The nuclear polyhedrosis virus of the alfalfa looper Autographa californica contains a double-stranded, circular DNA genome. Fourteen scientists agreed to accept an orientation of this circular genome with respect to a physical map of the restriction endonuclease cleavage sites.     

Isolation and Characterization of Sendai Virus Temperature-Sensitive Mutants

Ten temperature-sensitive mutants of Sendai virus, a paramyxovirus, were isolated and partially characterized. The mutants replicated in chicken embryo lung cells at 30 C, but not at 38 C; wild-type virus grew equally well at both temperatures. Complementation tests divided the mutants into seven groups. Six groups synthesized neither infectious virus nor RNA when incubated at 38 C from the beginning of infection. Temperature shift-up experiments demonstrated that three of these complementation groups were blocked in early steps required for RNA synthesis, but these gene functions were not needed throughout the replicative cycle. In contrast, the other three RNA-negative complementation groups were defective throughout the replicative cycle in functions required for virus-specific RNA synthesis. Only one mutant, which complemented all of the above, synthesized RNA but not infectious virus when placed at 38 C; the hemagglutinin of this mutant functioned only at the permissive temperature.     

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.     

In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs

A preliminary translational map of the Autographa californica genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected Spodoptera frugiperda cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of l-[(3)H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19 A. californica nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with l-[(3)H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.     

Interspersed Homologous DNA of Autographa californica Nuclear Polyhedrosis Virus Enhances Delayed-Early Gene Expression

The five regions of homologous DNA which are interspersed in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus increased the expression of a delayed-early gene of this virus. Although this activity was first observed as a 10-fold trans effect, the homologous region 5 (hr5) enhanced the expression of linked genes 1,000-fold. The hr5 enhancer also exhibited the other characteristics associated with viral enhancer elements, including orientation independence and the abilities to function at a distance from the linked promoter, to regulate heterologous promoters, and to increase the number of RNA polymerase molecules transcribing the linked genes. The expression of the immediate-early regulatory gene was not enhanced by cis-linked hr5, although the enhancer function may require the immediate-early regulatory gene product. The hr5 enhancer was relatively insensitive to competition by an excess of enhancer molecules. The nucleotide sequence of hr5 revealed two different conserved repeats separated by nonhomologous DNA. Deletion analysis of the hr5 enhancer indicated that a 30-base-pair inverted repeat was essential for enhancer function.     

Occluded and Budded Autographa californica Nuclear Polyhedrosis Virus: Immunological Relatedness of Structural Proteins

The immunological relatedness of the structural proteins of the budded and occluded phenotypes of Autographa californica nuclear polyhedrosis virus was examined by reciprocal immunoblotting and by in situ peroxidase-antiperoxidase staining of virus-induced cell surface and intracellular antigens with antisera to both phenotypes. The molecular weights (MWs) of major structural proteins of both phenotypes that reciprocally cross-reacted were 92,500, 78,000, 62,500, 54,000, and 42,000. A highly immunogenic, major structural protein of the occluded phenotype of 46,000 MW was not recognized by antiserum to the budded phenotype, and a major structural protein of the budded phenotype, 48,000 MW, was not recognized by antiserum to the occluded phenotype. Both the budded and occluded phenotypes contained a protein of 33,500 MW that comigrated with polyhedrin (the matrix protein) and reacted with antiserum and monoclonal antibody to polyhedrin. Evidence was obtained for the apparent antigenic relatedness of proteins of different MWs from the budded and occluded phenotypes, possibly indicative of different processing of some proteins for the two phenotypes. Antiserum to the occluded phenotype recognized virus-induced cell surface antigens, indicating antigenic similarities between the occluded phenotype and envelope proteins of the budded phenotype. Antiserum to the budded phenotype recognized viral proteins produced before the appearance of cytopathic effect, whereas antiserum to the occluded phenotype did not.     

Mapping the Mutation Site of an Autographa californica Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant

A polyhedron morphology mutant of Autographa californica nuclear polyhedrosis virus, designated M5, was compared with wild-type virus by genotypic analysis with EcoRI, BamHI, HindIII, SstI, and SmaI restriction endonucleases. M5 DNA revealed several alterations relative to the wild-type pattern: (i) EcoRI fragment I was 400 base pairs larger; (ii) BamHI fragment F was missing; (iii) HindIII fragment F was 400 base pairs larger; (iv) an extra restriction fragment was obtained with both HindIII and SmaI; and (v) SstI fragment G was 400 base pairs larger. M5 virions contained two size classes of circular DNA, one of 100% of the wild type and one of about 58% of the wild-type molecule. A revertant of M5, designated M5R, was isolated on the basis of polyhedron morphology. The genome of M5R contained the insertion of DNA in EcoRI fragment I and in HindIII fragment F, but was similar to the wild type in its other restriction fragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptide smaller in size than the wild type or M5R.     

Effects of Temperature and pH on Survival of Free Nuclear Polyhedrosis Virus of Autographa californica

The effects of temperature and low pH on replication and survival of nonoccluded Autographa californica nuclear polyhedrosis virus were investigated. No virus replication or formation of polynuclear inclusion bodies occurred at 37°C. The virus was immediately inactivated upon exposure to pH 2.0 and was inactivated within 1 h at pH 4.0. The virus titer slowly declined, a 3-orders of magnitude reduction in virus titer, at pH 5.0 during a 4-h exposure. Virus survival at pH 6.0 was equal to that of the control in cell culture medium 199 MK (pH 7.12).     

Complete Sequence and Enhancer Function of the Homologous DNA Regions of Autographa californica Nuclear Polyhedrosis Virus

The nucleotide sequence of the five regions of homologous DNA in the genome of Autographa californica nuclear polyhedrosis virus DNA was determined. The homology of repeated sequences within a region was 65 to 87%, and the consensus sequences for each region were 88% homologous to each other. Sequences proximal to the EcoRI sites were most conserved, while the distal sequences were least conserved. The EcoRI sites formed the core of a 28-base-pair imperfect inverted repeat. All homologous regions functioned as enhancers in a transient expression assay. A single EcoRI minifragment located between EcoRI-Q and -L enhanced the expression of 39CAT as efficiently as the regions containing numerous EcoRI repeats did.     

Isolation and Preliminary Characterization of Temperature-Sensitive Mutants of Influenza Virus

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10⁻³ or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.