A redox-sensitive cysteine in Zta is required for Epstein-Barr virus lytic cycle DNA replication.
Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.
Wang
Pu
P
The Wistar Institute, Philadelphia, PA 19104, USA.
Day
Latasha
L
Dheekollu
Jayaraju
J
Lieberman
Paul M
PM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics; D015854:Up-Regulation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D014779:Virus Replication
C063676:BZLF1 protein, Herpesvirus 4, Human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D012694:Serine; D003545:Cysteine