Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo

Abstract

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue, with a size equivalent to only 22 % of that of the largest orthologues. The present study focused on determining whether BoHV-4 ORF73 is a bona fide gene and investigating whether it is essential for latency, as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early polycistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by the wild type and revertant recombinants. Together, these results demonstrate that, despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology. INTRODUCTIONGammaherpesviruses, like all viruses in the family Herpesviridae, exhibit two distinct phases in their life cycle: lytic replication, characterized by a transcription programme in which immediate-early (IE), early (E) and late (L) genes are expressed successively; and latency, characterized by the maintenance of the viral genome as a nonintegrated episome and the expression of a limited number of viral genes and microRNAs (Cai et al., 2005;Roizman & Pellett, 2007;Sarid et al., 1998;Weck et al., 1999). Upon reactivation, latency shifts to lytic replication.The gammaherpesvirus human herpesvirus 8 (HHV-8, also termed KSHV for Kaposi's sarcoma-associated herpesvirus), is the prototype of the genus Rhadinovirus. During latency, HHV-8 expresses three main ORFs: ORF71, ORF72 and ORF73 (Sarid et al., 1998(Sarid et al., , 1999. ORF71 and ORF72 encode viral homologues of cellular FLIP (Thome et al., 1997) and D cyclin (Li et al., 1997), respectively. ORF71 and ORF72 are expressed together with ORF73 as a polycistronic mRNA (Dittmer et al., 1998;Talbot et al., 1999). Whilst some rhadinoviruses encode orthologues of ORF71 and/or ORF72, all rhadinoviruses encode an ORF73 orthologue. For example, bovine herpesvirus 4 (BoHV-4) encodes orthologues of ORF71 and ORF73, whereas ORF72 is absent (Zimmermann et al., 2001 pORF73 is the latency-associated nuclear antigen 1 (LANA-1) of HHV-8. LANA-1 associates with mitotic chrom...

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle

“…S10C). Absence of virus growth defect in culture was in line with some reports showing that γ-herpesvirus ORF73 is not essential for virus replication in vitro (21)(22)(23)(24), although MuHV-4 or Rhesus monkey rhadinovirus showed lytic replication deficit under certain conditions or enhancement, respectively (25,26). To further address the effect of ORF73 deletion on virus replication in vivo, we used the luciferase + WT AlHV-1 (247N-luc + ) BAC plasmid (15) to produce a 247N-luc + -73 null recombinant virus (SI Appendix, Fig.…”

“…γHV-68 ORF73-deficient virus displayed substantial growth deficit in murine fibroblasts at very low moi (25), and ORF73 disruption in Rhesus monkey rhadinovirus generated a highly lytic virus strain (26). Other than these two examples, most of the ORF73-deficient γ-herpesviruses tested so far have not shown any growth defect in vitro (21)(22)(23)(24)34). These reports suggest that ORF73 might affect viral growth in cell culture only under certain specific conditions.…”

“…The genome maintenance protein encoded by ORF73 in γ-herpesvirus tethers the viral episome to cellular chromosomes during cell division and therefore, is essential for virus persistence in vivo (22)(23)(24). One of the theories to explain MCF induction was that AlHV-1 infection could create an autoimmune-like environment, resulting in the trans activation and proliferation of uninfected T cells.…”

“…KSHV LANA is also able to up-regulate survivin expression and promote B-lymphoma cell proliferation (44). AlHV-1 encodes the largest genome maintenance protein described to date (24). It is, therefore, possible that AlHV-1 ORF73 might similarly have prooncogenic functions that could ultimately be involved in the triggering of CD8 + T-cell proliferation observed in MCF.…”

Herpesvirus bovino 4 (BoHV-4): aspectos generales de su biología y situación en la República Argentina

“…Se ha demostrado que la supresión del ORF 73 no afecta la capacidad del virus para replicar in vitro, pero su ausencia impide la producción de la infección latente en el modelo conejo. Esta propiedad derivada de la deleción del ORF 73 podría ser de utilidad en el uso del BoHV-4 como vector en la formulación de vacunas 84 .…”