Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) . We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-⌬UL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.Herpesvirus particles are characterized by the presence of four morphologically differentiable components: the inner nucleoprotein core with double-stranded genomic DNA; the icosahedral capsid shell; an amorphous material of protein called the tegument; and an envelope of host cell-derived lipids containing virus-encoded (glyco)proteins (26). Assembly of herpesvirus capsids occurs in the nuclei of infected cells. Intranuclear capsids acquire a primary envelope by budding through the inner nuclear membrane, resulting in perinuclear enveloped virions (13, 14, 26). These virions have been shown to be biochemically and ultrastructurally different from mature extracellular virus particles. In particular, perinuclear pseudorabies virus (PrV) virions contain the UL34 protein, which has been shown to be required for primary envelopment of herpes simplex virus type 1 (HSV-1) (27) as well as PrV (19). This protein is absent from intracytoplasmic or extracellular mature PrV virions. In contrast, the UL49 tegument protein is present in mature virions but absent from perinuclear virus particles (19). Therefore, the protein compositions of perinuclear immature and of intracytoplasmic and ex...

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-⌬UL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.The herpesvirus virion is a complex structure that contains at least 30 different virally encoded proteins. More than 15 viral proteins constitute the tegument that surrounds the herpesvirus capsid. The tegument itself is bounded by a cell-derived lipid bilayer envelope which contains more than 10 viral (glyco)proteins (28,35). Although the formation of capsids and the involved viral components has been delineated in some detail (41), the molecular mechanisms that drive assembly of the viral tegument and envelope during herpesvirus morphogenesis are still largely unknown. Initially, it had been postulated that herpesvirus virions are fully assembled in the nucleus, acquiring their final envelope by budding at the inner nuclear membrane. They should then be transported through the secretory pathway as complete virus particles (34). In this model, all envelope glycoproteins have to be present in the inner nuclear membrane and all tegument proteins have to be assembled onto the nascent virus particle in the nucleus. During the last few years, data have accumulated showing that capsids which bud at the inner nuclear membrane lose their primary envelope by fusion with the outer nuclear or the contiguous endoplasmic reticulum membrane and acquire a final envelope and tegument in the cytoplasm by budding into vesicles derived from the trans-Golgi network that contain mature viral glycoproteins (reviewed in reference 29). This envelopment-deenvelopment-reenvelopment mechanism revived investigations on the localization and site of addition of tegument proteins to the nascent virus par...

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