Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 K-bZIP modulates latency-associated nuclear protein-mediated suppression of lytic origin-dependent DNA synthesis.
The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.
Rossetto
Cyprian
C
Department of Microbiology and Immunology and the Cell and Molecular Biology Graduate Program, University of Nevada-Reno, Reno, Nevada 89557, USA.
Yamboliev
Irena
I
Pari
Gregory S
GS
D000956:Antigens, Viral / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods; D016213:Cyclins / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis*; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D000956:Antigens, Viral; D050976:Basic-Leucine Zipper Transcription Factors; D016213:Cyclins; D004279:DNA, Viral; C483104:K cyclin protein, Herpesvirus 8, Human; D009687:Nuclear Proteins; D014764:Viral Proteins; C109540:latency-associated nuclear antigen