The human herpes virus-8 (HHV-8) has a protein that appears to allow it to tether its genome to human chromosomes during cell division, ensuring that the virus is passed on from one generation of cells to the next during the phase of silent infection.
The finding, by a team of researchers in Boston, comes just as another international team, headed by scientists in the U.K., provided strong evidence to support the hypothesis that HHV-8 is the cause of Kaposi¿s sarcoma (KS). It could lead to therapies for KS and other cancers associated with HHV-8 infection.
It may also one day make it possible to overcome one of the biggest obstacles facing those trying to develop gene therapy: ensuring that a gene delivered to one cell is passed on to daughter cells during cell division.
Without such a method, therapeutic genes would have a life span only as long as the cells they initially entered.
Kenneth Kaye, assistant professor at Harvard Medical School in Boston, told BioWorld International: ¿One important potential application of our finding is in gene therapy. This finding should allow for persistence in a cell of any DNA sequence of interest, in the form of an extrachromosomal episome, or plasmid.¿
The study by Kaye, together with Mary Ballestas and Pamela Chatis, focused on the role of one of HHV-8¿s proteins, called latency-associated nuclear antigen (LANA). As the name suggests, this protein is manufactured by the virus during latent infection. This is the phase during which the virus silently replicates without killing its host cell, and usually without being detected by the immune system.
Kaye¿s study is complemented by that of Chris Boshoff, Glaxo Wellcome fellow and senior lecturer in the departments of Oncology and Molecular Pathology at University College London, in London. He is working with colleagues in London, Israel, France, Belgium and Finland who have developed an antibody capable of detecting LANA. Their work showed LANA was present in three different tumors: KS, multicentric Castleman¿s disease (an aggressive lymphoproliferative disease) and primary effusion lymphoma (PEL).
Boshoff told BioWorld International: ¿We showed that LANA is present in three different types of cells in these tumors. Now that we know one of the functions of this protein, from the study by Kaye and his colleagues, we know that every cell that has the virus in it will have expression of this protein, because it plays an essential role in maintaining the virus¿ genome.¿
The study by Ballestas and others is published in the April 23, 1999, issue of Science in a paper titled, ¿Efficient Persistence of Extrachromosomal KSHV DNA Mediated by Latency-Associated Nuclear Antigen.¿ The study by Boshoff¿s team is published in the April 23, 1999, Proceedings of the National Academy of Sciences in a paper titled: ¿Distribution of human herpesvirus-8 latently infected cells in KS, multicentric Castleman¿s disease, and primary effusion lymphoma.¿
For their research, Kaye and colleagues used cells from primary effusion lymphomas. This is a rare type of cancer that occurs most often in people with AIDS. It causes fluid to collect in a body cavity, such as that between the lungs and the chest wall, and the malignant cells grow in suspension in this fluid. Studies have shown that HHV-8 ¿ which also is known by the name KS-associated herpesvirus, or KSHV ¿ is present in these cells, and that LANA is expressed in their nuclei. LANA is similarly found in the malignant cells of KS lesions.
Kaye¿s team used three different strains to identify the dividing human chromosomes in the PEL cells, the viral DNA, and the LANA protein. He said: ¿We were able to see that the viral DNA, which is in the form of an episome or plasmid ¿ an extrachromosomal circle of DNA ¿ co-local ised with LANA in dots, and that these dots were found on the human chromosomes.
This was consistent with a model in which, during cell division, LANA tethers the viral genome to the chromosomes, so that the chromosomes carry the tethered genomes to each daughter cell.¿
To test this model, they took cells that were not infected with the virus, and manipulated them so that they expressed the LANA gene but no other viral genes. ¿We found that these cells were able to allow the persistence during many cell divisions of episomes containing a certain viral sequence. Cells without LANA were not able to permit the persistence of this DNA as an episome,¿ Kaye said.
Kaye and his colleagues believe LANA may bind the essential viral sequence the plasmid needs in order to persist as an episome. They are now embarking on studies to find out exactly what this sequence is, and how LANA binds to the human chromosomes.
The finding could be applied to gene therapy, Kaye predicted. ¿Any gene of interest could be inserted into a plasmid which expresses LANA and contains the essential sequence of DNA required for LANA-mediated episome persistence,¿ he said. ¿This DNA would carry the gene of interest and persist in replicating cells as an episome.
¿This discovery also provides a potential basis for therapy,¿ Kaye said. ¿If one could interfere with this function of LANA, then one should be able to abort HHV-8 infection.¿ He predicted that screening compounds to find those that are active against LANA could be carried out quite easily. The group is currently seeking partners to expand its work in this field.
Boshoff¿s group has developed an antibody against LANA. It will be marketed commercially by ABI Inc., of Columbia, Maryland. Boshoff predicted that the antibody would be of great use to anyone wanting to confirm the presence or absence of HHV-8 infection in a tissue.
The results reported in the PNAS paper show that HHV-8 is present in immunoblast cells of the B cell lineage ¿ essentially immature B cells ¿ in multicentric Castleman¿s disease, and in immature plasma cells in PEL. The group also looked at which types of cells in KS were infected with HHV-8.
¿We have shown, for the first time,¿ Boshoff said, ¿that the virus is present in immature endothelial cells, cells that probably belong to the lymphatic system, and that, from the beginning, the virus is present in the type of cell that eventually gives rise to KS. Our data strongly support the hypothesis that HHV-8 is the cause of KS.¿ Their study also ruled out the involvement of HHV-8 in multiple myeloma, prostate cancer and angiosarcoma.
Boshoff and colleagues are currently using the antibody against LANA to further investigate the biochemical role of this protein. n