Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs

J Virol. 2017 Sep 12;91(19):e00992-17. doi: 10.1128/JVI.00992-17. Print 2017 Oct 1.

Abstract

Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

Keywords: Kaposi sarcoma-associated herpesvirus; LANA; gammaherpesvirus; latency-associated nuclear antigen; latent infection; lytic infection; murid herpesvirus 4; murine gammaherpesvirus 68; small animal model.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3T3 Cells
  • Animals
  • Antigens, Viral / biosynthesis
  • Antigens, Viral / genetics*
  • Cell Line
  • Female
  • Gene Knock-In Techniques
  • HEK293 Cells
  • Herpesvirus 8, Human / genetics
  • Herpesvirus 8, Human / growth & development*
  • Humans
  • Immediate-Early Proteins / genetics*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Nuclear Proteins / biosynthesis
  • Nuclear Proteins / genetics*
  • Promoter Regions, Genetic / genetics
  • Rhadinovirus / genetics
  • Rhadinovirus / growth & development*
  • Rhadinovirus / metabolism
  • Trans-Activators / genetics*
  • Virus Latency / genetics*

Substances

  • Antigens, Viral
  • Immediate-Early Proteins
  • Nuclear Proteins
  • Rta protein, Human herpesvirus 8
  • Trans-Activators
  • latency-associated nuclear antigen