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

Abstract

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. Nuclear polyhedrosis viruses are members of subgroup A in the family Baculoviridae. A distinctive feature of these invertebrate DNA viruses is the occlusion of many enveloped virus particles in a large paracrystalline inclusion, which is referred to as a polyhedron. About 95% of the polyhedron mass is composed of a single polypeptide having a molecular weight of approximately 30,000, which is referred to as poly

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses

“…Labeling of infected-cell-specific proteins. Virus-induced proteins were identified in S. frugiperda cells infected with 10 PFU per cell of each virus isolate by pulse-labeling infected and mock-infected cell cultures with [3H]leucine as described by Vlak et al (43). Cultures were pulsed for 3-h intervals at 9, 15, and 24 h postinfection.…”

Effect of baculovirus infection on the mRNA and protein levels of the Spodoptera frugiperda eukaryotic initiation factor 4E

“…During a baculovirus infection cellular protein synthesis is shut off (Vlak et al , 1981). The aim of this research was to determine whether host shut off mechanisms in baculovirus-infected lepidopteran insect cells involved the mRNA cap-binding protein eIF4E.…”

Cloning and analysis of cDNAs encoding the hypusine‐containing protein eIF5A of two lepidopteran insect species

“…We are interested in post‐transcriptional aspects of gene expression in lepidopteran insect cells and in the alterations that occur in the translational machinery following a baculovirus infection. Upon a baculovirus infection the synthesis of host proteins is shut off while late baculovirus genes are still highly expressed ( Vlak et al ., 1981 ). To understand this phenomenon, detailed information is needed on translation initiation factors and the control mechanisms functioning in insect cells.…”