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Virus Ensures Persistence by Tethering Genome to Host DNA

The Epstein-Barr virus (EBV) genome—and possibly other extrachromosomal DNA molecules—apparently is tethered by means of a specialized protein to the host cell chromosome. This linkage helps to guarantee that the viral DNA is faithfully replicated and maintained in appropriate copy numbers whenever host cells divide, and it may represent a common mechanism for "enhancing the replication and transmission of extrachromosomally replicating viruses’’ and perhaps other extra bits of DNA, according to Geoffrey M. Wahl of the Salk Institute in La Jolla, Calif., and his collaborators. These findings thus illuminate some long-standing mysteries surrounding DNA replication and segregation, and could provide insights for those developing antiviral agents and vectors for use in gene therapy.

Like many cell-adapted viruses, latently infected EBV replicates synchronously with its host cell each cycle—doing so without relying on DNA enzymes of its own or a centromere, which is the structure within cellular chromosomes that distributes them evenly from parent to daughter cells. The virus depends on comparably efficient DNA strand separation, or segregation, to avoid being lost following mitosis. Yet it does so using a distinct mechanism, according to Wahl and his collaborators, who describe their findings in Molecular and Cellular Biology (21:3576-3588, 2001).

A key component of the viral genome separation apparatus is an EBV-specified protein, called nuclear antigen 1 (EBNA-1), that is required for DNA replication. However, its role in replication eluded researchers altogether until several years ago, when Michelle Calos and her collaborators at Stanford University in Stanford, Calif., implicated EBNA-1 as somehow retaining viral DNA molecules within the nucleus of host cells. Meanwhile, other researchers suggested that EBNA-1 fastened the viral genome to host chromosomes in the nucleus, juxtaposing the EBV genome with the host replication apparatus and presumably enabling it to replicate the nearby viral genome.

``But that was based on indirect analysis,’’ Wahl says. ``Nobody had been able to look directly at the viral molecule, the EBNA-1 protein, and the chromosome of the cell at the same time, for technical reasons.’’ However, Teru Kanda, then a fellow in Wahl’s lab and now at Hokkaido University in Sapporo, Japan, used the green fluorescent protein (GFP), which is derived from jellyfish and glows brilliantly when illuminated with light of the appropriate wavelength, to visualize where and what EBV plasmid and EBNA-1 protein are doing in host cells.

"We confirmed that EBNA-1 really does mediate tethering [of EBV DNA to the host cell] chromosome because we can see the [EBV] plasmids as green dots, linked to the EBNA-1 protein, which is linked to the chromosome,’’ says Wahl. "There seems to be a [functional] link between tethering and replication of the viral plasmid molecule.’’ For instance, deleting the portion of EBNA-1 that is used to tether the plasmid to mitotic chromosome blocks viral replication. Biochemical analyses reveal that the same deletion disrupts the association of EBNA-1 protein with interphase chromatin (decondensed chromosome in interphase nuclei).

The result implies that EBNA-1-mediated chromatin association of EBV plasmids increases both the replication efficiency during S phase and the segregation efficiency during mitosis. "So EBNA-1 is involved in replication not because it has an intrinsic catalytic activity that contributes to replication, but because it moves these extrachromosomal molecules to the chromosome (interphase chromatin), where they can access the replication machinery,’’ Wahl says.

The notion that EBNA-1 might fasten viral DNA to a cellular chromosome resonates with research dating back several decades indicating that "double minute chromosomes’’ (DMs), a class of extrachromosomal circles of DNA, may also be linked to host chromosomes, according to Wahl. Moreover, because some DMs carry oncogenes, these recent EBV-related findings could well have broader significance. "If we knew how DMs replicate and segregate, we might be able to design drugs to interfere with these critical processes,’’ he says. "If we could effect loss of DMs from cells using such agents, our previous work and that of others strongly suggests that this would cause the cancer cells to die.’’

The findings likely apply to other mammalian viruses, according to Wahl. "The tethering of viral genomes to host chromosomes appears to be a common aspect of the life cycle of DNA viruses with a latent infection phase,’’ he points out. "These include other gamma-herpesviruses, such as Kaposi’s sarcoma-associated herpesvirus, and herpesvirus saimiri, as well as bovine papillomavirus.’’ Because these viruses appear to possess the molecular machinery needed for tethering their genomes to host chromosomes by similar means, Wahl and his collaborators say that "chromosome tethering may be a common mechanism for enhancing the replication and transmission of extrachromosomally replicating viruses into daughter nuclei.’’

These results could prove relevant to researchers developing vectors for use in gene therapy, according to Elliot Kieff of Harvard Medical School in Boston, Mass., who also is studying viral genome tethering and who calls Wahl’s work "elegant.’’ Kieff and his colleagues reported early this year that histone H1 or HMGI, another basic protein that binds to mammalian chromosomal DNA, can partly substitute for EBNA-1 in its viral genome-tethering role. The amino terminus of EBNA-1 enables the viral plasmid to persist within the host cell nucleus, Kieff says, and that capacity to maintain DNA from an outside source within a mammalian cell is a critical part of what is needed for vectors being designed for use in gene therapy.

David Holzman
David Holzman is a freelance science writer in Lexington, Mass.