A Novel Class of Growth Factors Related to Herpesviruses

Konval ina I . , J . Ga‰perik, F. Golais : A Novel Class of Growth Factors Related to Herpesviruses. Acta Vet. Brno 71, 2002: 29-36. In the last decade a novel class of growth factors related to the herpesvirus group was characterized which substantially differ from other virus-related growth factors which represent viral versions of known cellular growth factors. This novel class cannot be related to any known cellular products. They have an ability to transform non-transformed cells in vitro and to suppress the transformed phenotype of transformed cells. The biological activities of these factors could be neutralized not only by antisera to corresponding virus, but, as shown with two of these factors, also by some monoclonal antibodies directed against viral gB glycoprotein. Furthermore, studies with some mutants in gene for gB revealed that this gene might be involved in growth factor synthesis. Another characteristic making them different from other growth factors is their low molecular weight (< 103) and their component character, they consist of 2 or 3 active components. In vivo studies showed that they may influence embryonic or postembryonic development of some animals, e.g. mice, rats or fish. Some unusual properties, e.g. extremely high titres of their biological activity demonstrated in cell cultures, or enhancement of this activity following temperature and urea treatment, or UV irradiation render them attractive for further studies indicating their peculiar structure, which is still obscure. Pseudorabies virus growth factor, biological and physicochemical properties, transformed phenotype Some poxviruses and herpesviruses have been shown to code for secretory proteins with structural similarity to cellular growth factors or similar polypeptides such as cytokines and chemokines. Vaccinia virus, myxoma virus, variola virus and fibroma virus encode a polypeptide which is structually homologous to both epidermal growth factor and alpha transforming growth factor (Stroobant et al. 1985; Porter and Archard 1987; McFadden et al. 1995; reviewed by Kontsek and Kontsekova 2000), or orf virus encode a factor resembling vascular endothelial growth factor (Lyt t le et al. 1994). Some herpesviruses acquired the ability to code for homologs of cytokines. Cells infected with human herpesvirus 8 (HHV-8 or Kaposi’s sarcoma-associated herpesvirus, KSHV) secrete protein similar to human interleukin 6 (IL-6) (Moore et al. 1996; Nicholas et al. 1997). BCFR1 gene product of Epstein-Barr herpesvirus has 89% amino acid identity with mature human IL-10 (Moore et al. 1990) and similarly equine herpesvirus type 2 codes for protein possessing high homology to human and murine IL-10 (Rode et al. 1993). IL-10 is also encoded by orf poxvirus (Fleming et al. 1997). Some herpesviruses code for chemokines (McDonald et al. 1997; Dairaghi et al. 1998; Zou et al. 1999). For all these viral products mentioned above, the term virokines was coined (Kotwal 1999; Kontsek and Kontsekova 2000). Finally, some gamma herpesviruses such as Herpesvirus saimiri, KSHV, or murine herpesvirus 68 produce D-type cyclin homologs (reviewed Laman et al. 2000). ACTA VET. BRNO 2002, 71: 29–36 Address for correspondence: Doc. RNDr. Franti‰ek Golais, CSc. Department of Microbiology and Virology Faculty of Natural Sciences, Comenius University Mlynska Dolina B-2, 842 15 Bratislava, Slovak Republic Phone: +421 2 6029 6487 Fax: +421 2 6029 6686 E-mail: gdais@fns.uniba.sk http://www.vfu.cz/acta-vet/actavet.htm All these products represent viral versions of cellular growth factors, cytokines, or cyclins. Virus genes coding for them were captured from the host genome during evolution of viruses (Spriggs 1994). In the last decade a new class of growth factors related to herpesviruses appeared whose characteristics are considerably different from all known virokines. The aim of this paper is to summarize the present knowledge about these growth factors. History and background Although the direct role of some alpha herpesviruses in the formation of malignant tumours is questionable, if at all, there exists a body of evidence for their oncogenic potential in vitro (Rapp and Reed 1976; Hampar 1981), even though the mechanisms of transformation are still unknown (Gal loway and McDougal l 1981; 1990). Golais et al. (1985) described an original model of transformation. Human embryonic lung (HEL) cells infected with pseudorabies virus (PRV) at a low multiplicity of infection (0.001-0.01) and cultivated in the presence of antiviral antibodies and human leucocyte interferon yielded foci of morphologically transformed cells from which a stabile transformed cell line HPR1 could be derived. Subsequent studies revealed that when a crude extract of HPR-1 cells was added to HEL cells, morphological signs of transformation were observed and this effect could be removed, when extract was treated with anti-PRV antibodies (Golais et al. 1988). Further studies have shown, that not only extracts from transformed HPR-1 cells but also those from PRV infected HEL cells devoid of infectious virus possessed such ability. Furthermore, when these extracts were added to transformed (e.g. HeLa) cells the repression of the transformed phenotype was observed (Golais et al. 1990). Similar results were achieved with herpes simplex virus type l (HSV-1 or herpes virus hominis type 1 – HHV-1) and HSV-2 (HHV-2), (Golais et al. 1992ab), as well as with some other herpesviruses (Ga‰perik et al. 1996). Isolation and basic characteristics of herpesvirus related growth factors Growth factors related to herpesviruses could be detected either in virus transformed cells, e.g. PRV related growth factor (PRGF), which was originally obtained from PRV transformed HPR-1 cells, or in cells infected with virus at low MOI and cultivated in conditions which are non-permissive for replication of virus. Such condition could be achieved during cultivation of infected cells at 41 °C or in the presence of DNA synthesis, e.g. phosphonoacetic acid (PAA). Cells infected with PRV or HSV-2 and kept at 41 °C for 4-5 days produced little or no virus and only small amounts of PRGF respectively HSGF-2 (growth factor related to HSV-2). The production of both PRGF and HSGF-2 was considerably enhanced when the temperature was shifted down to 37 °C. The reactivation of virus growth after temperature shift proceeded much more slowly than that of PRGF and HSGF-2 production, so that virus-free samples could be obtained. To remove trace amounts of virus, the media containing growth factors were acidified to pH 3, kept for 72 h at +5 °C, and afterwards their pH was raised again to neutrality (Golais et al. 1992a). PAA was shown to completely inhibit the synthesis of PRGF (Golais et al. 1990). Cells infected with PRV and HSV-2 were cultivated in the presence of PAA for 4-5 days, then the medium containing PAA was removed and replaced with intact medium. The production of both PRGF and HSGF-2 started 24 h following PAA removal. In both cases the production of both PRGF and HSGF-2 was considerably enhanced when human leucocyte interferon (IFN) was added after temperature shift or PAA removal, however, it was completely blocked in the presence of methylation inhibitor 5-azacytidine (Golais et al. 1992a). Some cells are non-permissive for replication of some herpesviruses even in normal cultivation conditions, the cells of human origin represent such system for PRV (Golais 30