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Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of varying amounts of twenty or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions within extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein: a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36 whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.IMPORTANCENeuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals, but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument and with it, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes.