Herpesvirus Replication Compartments Originate with Single Incoming Viral Genomes

Department of Molecular Biology and the Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
mBio (Impact Factor: 6.79). 10/2011; 2(6). DOI: 10.1128/mBio.00278-11
Source: PubMed


Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments.

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    • "The first steps of herpesviruses virion assembly occur in the nucleus where the replicated DNA must be embedded into the preformed capsids. It is quite well established that the replication starts at specific spots that increase over time, in number and mainly in size, until they finally fuse to give rise to what has been called the " replication compartment " (RC) (de Bruyn Kops and Knipe, 1994; Kobiler et al., 2011; Quinlan et al., 1984). HSV-1 capsid assembly is well documented (for review see Cardone et al., 2012; Homa and Brown, 1997). "
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    • "Recruitment of HSV Pol (UL30/42) requires the presence of an active primase (Carrington-Lawrence and Weller 2003), and once this occurs, small replication compartments form, at which time ICP8 foci appear to merge with ICP4/27 foci (Livingston et al. 2008). Recent studies that monitor the location of incoming genomes have indicated that each RC forms from a single genome and that only a limited number of incoming genomes are actively expressed and go on to be replicated (Kobiler et al. 2011). "
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