Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase α‐primase

Abstract

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase a-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase a-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.Keywords: DNA replication; initiation; DNA polymerase a-primase; species specificity; polyomavirus.Polyomavirus DNA replication has served as a model system to study eukaryotic DNA replication [1,2]. JC virus (JCV) belongs to the polyomavirus family and is the causative agent of progressive multifocal leukoencephalopathy in immunocompromised humans (reviewed in [3][4][5][6][7]). JCV exhibits a highly restricted host range and this species specificity appears to be governed by host encoded DNA replication factors as hamster glial cells, which support viral early gene transcription, nevertheless fail to replicate JCV DNA [8]. JCV is closely related to simian virus 40 (SV40) and to mouse polyomavirus (PyV), both of which show clear species specificities as lytic infection is limited to primate and mouse cells, respectively [9]. The species specificities of both SV40 and PyV are regulated at the level of initiation of DNA replication [10], a process that has been extensively studied both in vivo and in vitro owing to the development of cell-free DNA replication systems [2,[11][12][13][14][15][16].Polyomavirus DNA replication is carried out by the host cell machinery supplemented with a single essential viral protein, large T antigen (TAg), which recognizes and partially unwinds the viral replication origin, recruits host proteins such as replication protein A (RPA) and DNA polymerase a-primase, and functions as the replicative helicase [2,17,18]. Species specificity of both SV40 and PyV DNA replication can be reproduced in vitro using DNA carrying the viral core origin and purified replication enzymes [14,[19][20][21]. For both SV40 and PyV it has been clearly demonstrated that the host factor responsible for species specificity is DNA polymerase a-primase, which initiates DNA replication in all eukaryotes [19,[22][23][24][25][26][27]. DNA polymerase a-primase consists of four subunits with apparent molecular masses of 180, 68, 58 and 48 kDa of which the largest and smallest subunits are a DNA polymerase and a primase, respectively [28][29][30]. SV40 DNA replication in vitro was recently shown to require a functional interaction between the SV40 TAg and the C-terminus of the p180 subunit of human DNA polymerase a-primase [26].The genome of JCV is 69% homologous to that of SV40 and expresses an analogous set of proteins [31]. The core origins of r...

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

“…This suggests for the first time that the human Pol-prim complex is a speciesspecific factor for BKV DNA replication. This is in agreement with mPyV and SV40 DNA replication, whereas it is contrary to JC virus (JCV) DNA replication (2,45,47). The presence of an inhibitory activity in mouse extracts (28) raises the question of whether mouse Pol-prim could be the possible inhibitor of BKV DNA replication in mouse extracts.…”

“…However, they do not give any additional information about the inhibitory factor(s) found in mouse cell extracts. This activity is specific for BKV (28), and it has not yet been reported for another viral system, i.e., not JCV, mPyV, or SV40 (28,45,47). As we demonstrated earlier, the inhibitory effect is greater when mouse extract is added during the unwinding step of DNA replication (28).…”

Restriction of Human Polyomavirus BK Virus DNA Replication in Murine Cells and Extracts

“…It is noteworthy that JCV TAg is reported to support replication of DNAs with its cognate origin in murine extracts (89). These differences also might be reflected by the origin selectivity of the virus TAg proteins for their cognate origins.…”

“…To define the cause of the lack of BKV replication in murine cells, an in vitro BKV TAg-dependent DNA replication system was established. Since insect cells efficiently express polyomavirus TAg proteins capable of supporting cellfree DNA replication (9,68,79,89,93,94,103), BKV TAg was expressed from baculovirus vectors in High Five insect cells and purified (data not shown). Archetype BKV, which is derived from human patients but does not replicate in cultured cells, was used as the source of DNA for these experiments.…”

“…In addition to these shared properties, differences in replication between viruses have been reported to occur because of structural variation between viral core origins and TAg pro-teins (4,5,47,51,52); differences in TAg acetylation that either stimulate replication (109), or regulate TAg stability (73,84); selective interactions with the host p180 DNA Pol ␣-primase and p48 primase subunits (9,94) and with RPA (103); steps in replication beyond initiation (89); and modulation of replication by 5Ј and 3Ј cis-acting origin proximal sequences that alter activities of replication proteins and determine chromatin structure and intranuclear DNA localization (14,29,63,91,100,104,106,109). Such virus-and host-specific features may affect the outcomes of viral infection.…”