Relationship between vaccinia virus intracellular cores, early mRNAs, and DNA replication sites

Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed by the process of DNA replication. In the present study the quantitative and structural relationships between these distinct steps of VV morphogenesis were investigated. We show that viral RNA and DNA synthesis increases linearly with increasing amounts of incoming cores. Moreover, at multiplicities of infection that result in 10 to 40 cores per cell, an approximately 1:1 ratio between cores and sites of DNA replication exists, suggesting that each core is infectious. We have shown previously that VV early mRNAs collect in distinct granular structures that recruit components of the host cell translation machinery. Strikingly, these structures appeared to form some distance away from intracellular cores (M. Mallardo, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:3875-3891, 2001). In the present study the intracellular locations of the sites of early mRNA accumulation and those of the subsequent process of DNA replication were compared. We show that these are distinct structures that have different intracellular locations. Finally, we study the fate of the parental DNA after core uncoating. By electron microscopy, cores were found close to membranes of the endoplasmic reticulum (ER) and the parental DNA, once it had left the core, appeared to associate preferentially with the cytosolic side of those membranes. Since we have previously shown that the process of DNA replication occurs in an ER-enclosed cytosolic "subcompartment" (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001), the present data suggest that the parental DNA is released into the cytosol and associates with the same membranes where DNA replication is subsequently initiated. The combined data are discussed with respect to the cytosolic organization of VV morphogenesis.