- Volume 48, Issue 1, 1980

DNA replication complexes were purified from adenovirus type 5 (Ad5)-infected HeLa cells. DNA synthesis by these complexes in vitro was extremely sensitive to the inhibitors dideoxythymidine triphosphate, N-ethyl maleimide and p-hydroxymercuribenzoate. The bound DNA polymerase was released from the complexes by limited digestion with micrococcal nuclease. This released polymerase preferred poly(rA):(dT)12–18 as template over activated calf thymus DNA. These results are compatible with the major polymerase in the replication complex being of the γ class.

A focus-forming assay for the quantification of defective interfering (DI) particles of vesicular stomatitis virus (VSV) is described. This assay is based on the procedure described for lymphocytic choriomeningitis (LCM) virus by Popescu et al., (1976). Under appropriate conditions this focus-forming assay can quantify fewer than 100 DI particles/ml in a preparation containing a large number of infectious particles.

Cell ghosts were formed by hypotonic haemolysis and subsequent isotonic re-sealing of human erythrocytes in the presence of adenovirus type 2 DNA. The ghosts entrapped virus DNA with an efficiency which depended on the salt concentration employed during haemolysis and on the concentration of the DNA itself. The entrapped DNA was largely protected from digestion by deoxyribonuclease I and could be recovered intact by phenol extraction. Erythrocyte ghosts containing 32P-adenovirus type 2 DNA were fused with KB cells during brief treatment with polyethylene glycol (PEG). Following fusion, counts equivalent to an average of 25 molecules of labelled DNA were micro-injected and transported to each K B cell nucleus. Less than ¼ as many DNA counts were recovered from nuclei when PEG treatment was omitted. A direct immunofluorescent assay demonstrated virus replication in some cells following their fusion with DNA-containing ghosts. The efficiency of transfection was considerably lower than that expected from the large number of successfully micro-injected DNA molecules. This suggests that most of the micro-injected DNA molecules were degraded before a successful infection could be completed.

Recombination experiments involving a series of two-factor crosses have been done with ts mutants of the baculovirus, Autographa californica nuclear poly-hedrosis virus (NVP). Six mutants which fail to produce non-occluded virus at 33 °C (NOV mutants) have been ordered on a linear genetic map.

A recombinant between simian rotavirus, simian agent 11 (SA-11) and bovine rotavirus, neonatal calf diarrhoea virus (NCDV), was obtained by mixed infection of MA-104 cells with NCDV and u.v.-irradiated SA-11 virus, and isolation of a plaque formed in the presence of anti-NCDV serum. The genome of the recombinant contained dsRNA segments 4, 5 and 10 derived from SA-11 virus and segments 1, 2, 3, 6 and 11 derived from NCDV, and segments 7, 8 and 9 of undetermined origin. Polypeptides VP4, VP5, VP7a, VP7b, NCVP1 and NCVP3 were derived from SA-11 virus and polypeptides VP1, VP2, VP3, VP6, VP8, NCVP2a and NCVP2b from NCDV. Haemagglutination of the recombinant was inhibited and its infectivity neutralized by the antiserum against SA-11 virus but not by anti-NCDV serum.