Identification of a herpesvirus Saimiri cis-acting DNA fragment that permits stable replication of episomes in transformed T cells

Abstract

Herpesvirus saimiri is a lymphotropic herpesvirus capable of immortalizing and transforming T cells both in vitro and in vivo.Immortalized and transformed T cells harbor several copies of the viral genome as a persisting episome. The mapping of the cis-acting genetic segment (oriP) required for viral episomal maintenance is reported here. Viral DNA fragments that potentially contain oriP were cloned into a plasmid that contains the hygromycin resistance gene. After several rounds of subcloning followed by transfection, oriP was mapped to a 1.955-kb viral segment. This viral fragment permits stable plasmid replication without deletion or rearrangement as well as episomal maintenance without integration or recombination. The function of oriP depends on a trans-acting factor(s) encoded by the viral genome. The 1.955-kb viral segment includes a dyad symmetry region located between two small nuclear RNA genes and is located upstream of the dihydrofolate reductase gene homolog. Therefore, this oriP contains novel elements distinct from those of other DNA viruses.Animal viruses have provided useful systems for the study of DNA replication in eukaryotic cells. Characteristic features of the replication of latent genomes of DNA viruses are the presence of specific viral sequences that serve as origins of DNA replication and at least one specific viral protein whose direct interaction with these sequences is thought to be involved in the initiation of the replication process (11,14). These viral origins of replication may be models for the regulated replication of cellular chromosomes or, alternatively, adaptations that latent viruses evolved to use the normal cell cycle controls for maintenance of viral genomes.Herpesvirus saimiri (HVS) is the prototype of the lymphotropic ␥-2 subfamily of herpesviruses (46). The natural host of the virus is the New World primate squirrel monkey, which is normally persistently infected without causing any diseases (39). However, HVS infection of numerous other New World primates as well as New Zealand White rabbits results in rapidly proliferating tumors of T lymphoid cells (13,38,40). Cell lines established from infected animals show no signs of producing viruses after long-term culture (27,53). Moreover, human primary T cells can be transformed by group C strains of HVS to continuously proliferating T-cell lines with either the CD4 ϩ or CD8 ϩ phenotype (7,36). Immortalized T cells harbor the HVS viral genome as a persisting circular form in high copy number (7,27,36,53).HVS is closely related to Epstein-Barr virus (EBV), whose episomal replication has been studied in great detail. EBV episomal DNA replication starts from a fixed region of the viral genome, termed the origin of plasmid replication (oriP) (48, 56). The EBV oriP consists of a family of direct repeats (FR) and a dyad symmetry element (45). This cis-acting genetic element requires a virus-encoded protein, EBNA1 (for EBV nuclear antigen 1), in trans for episomal replication and persistence in latently infected cells (34, 57)....

Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus Saimiri

“…Probably C139 carries a further sequence divergence in an L-DNA region highly conserved among transforming and nontransforming virus strains. A possible origin of plasmid replication has been recently localized to a polymorphic region upstream of the dihydrofolate reductase gene in virus strain C484M (41).…”

Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

“…However, the FR of oriP is also a potent EBNA-1dependent enhancer of transcription, and EBNA-1 and the FR appear to be important for proper transcription from two EBV promoters that give rise to expression of genes required to immortalize B cells (10,33,39). Among the more distantly related gamma herpesviruses that are known to support autonomous replication of their viral genomes during latent infection, none appears to have a homolog of EBNA-1 or of oriP (2,5,24,40). It therefore seemed possible that the plasmid maintenance property of EBNA-1 and oriP could be functionally redundant, like the replication initiation function.…”

An Autonomous Replicating Element within the KSHV Genome

“…Additionally, depletion of LANA did not completely abolish the episomes from PELs suggesting the presence of LANA independent replication origins (Godfrey et al, 2005). Similarly, HVS lacking identified replication origins are capable of establishing latent infection (Kung and Medveczky, 1996). This suggests that these herpesviruses do not have an absolute requirement for a particular origin, although they may contain a dominant element.…”