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Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication
Abstract:
A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-form (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.5 degrees C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuIII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent, ts14 inhibited infectious virus production of H-1 or H-3, but not LuIII. LuIII-or H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influence RF DNA replication.
Insights
A new temperature-sensitive mutant, ts14, shows defective parvovirus replicative-form DNA synthesis. This defect is cis-acting and impacts viral DNA replication and protein synthesis, suggesting a regulatory sequence mutation.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Parvoviruses are small, single-stranded DNA viruses that replicate in the nucleus of host cells.
- Replicative-form (RF) DNA synthesis is a critical step in the parvovirus life cycle.
- Temperature-sensitive mutants are valuable tools for studying viral gene function.
Purpose of the Study:
- To isolate and characterize a temperature-sensitive mutant of H-1 parvovirus with defects in RF DNA synthesis.
- To investigate the genetic basis and functional consequences of the observed defect.
- To explore the interactions of the mutant with other parvoviruses.
Main Methods:
- Isolation and characterization of a temperature-sensitive mutant (ts14) of H-1 parvovirus.
- Assay of replicative-form (RF) DNA synthesis at permissive and restrictive temperatures.
- Complementation analysis using defective H-1 virus (DI-1) and wild-type H-3.
- Multiplicity-dependent inhibition assays for viral DNA and protein synthesis.
- Comparative physical mapping of viral DNA.
Main Results:
- The ts14 mutant exhibited significantly reduced RF DNA synthesis (3-7% of wild-type) at the restrictive temperature.
- Complementation analysis indicated that the RF DNA synthesis defect is cis-acting.
- ts14 demonstrated multiplicity-dependent inhibition of DI-1 and H-3 RF DNA synthesis, but not LuIII.
- Cross-interference was observed in viral protein synthesis, with ts14 inhibiting H-1 and H-3 infectious virus production.
- Physical mapping suggested higher homology between H-1 and H-3 than between H-1 and LuIII.
Conclusions:
- The ts14 mutant possesses a defect in a regulatory DNA sequence essential for H-1 parvovirus RF DNA replication.
- This mutation affects not only viral DNA synthesis but also viral protein synthesis and infectious virus production.
- The findings provide insights into the regulatory mechanisms governing parvovirus DNA replication and inter-viral interactions.