Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Richard W.P Smith,Claudia Steffen,Frank Grosse,Heinz-Peter Nasheuer

doi:10.1074/jbc.m201908200

Abstract

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase alpha-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase alpha-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase alpha-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase alpha depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase alpha-primase but rather on sequences in the C-terminal region of human p180.

Highlights

Polyomavirus DNA replication has been studied extensively owing to the ease with which viruses of this family, which include Simian Virus 40 (SV40) and Mouse Polyoma Virus (PyV), can be grown in cell culture and to the availability of an in vitro replication system which allows detailed investigation of the individual factors that play a role in the replication process [1,2]
Initiation of SV40 replication requires the formation of an active quaternary complex between DNA polymerase α-primase, TAg, RPA and the viral origin of replication
We concentrated on the functional interactions between SV40 TAg and DNA polymerase α-primase and, on the p180 subunit known to control SV40 species specificity [29]

Summary

Introduction

Polyomavirus DNA replication has been studied extensively owing to the ease with which viruses of this family, which include Simian Virus 40 (SV40) and Mouse Polyoma Virus (PyV), can be grown in cell culture and to the availability of an in vitro replication system which allows detailed investigation of the individual factors that play a role in the replication process [1,2]. SV40 and PyV are very similar with regard to DNA replication; their respective TAgs are 36% identical, have largely the same biochemical activities and recognise very similar pentanucleotide motifs at their replication origins which too exhibit great similarity [21] Both viruses show clear differences with respect to the host cells in which DNA replication can be initiated successfully. We show that the N-terminal 488 amino acids of human p180, comprising the TAg binding domain, are not responsible for the species specificity of DNA replication nor for efficient stimulation of DNA polymerase activity by TAg on ssDNA These results are supported by data obtained with surface plasmon resonance (BIAcore) which shows that SV40 TAg shows little difference in affinity for DNA polymerase α-primase from either human or murine origin

Materials and Methods

Discussion