Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E

Abstract

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By twodimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

“…Phosphorylation of lamins presumably leads to local dissolution of the lamina (12,21). Both proteins are also components of the primary virion but are absent from mature virus particles (8,10,11,22,23). In contrast, major tegument proteins of mature virions such as pUL36, pUL37, pUL46, pUL47, or pUL49 are not detectable in primary virions (24).…”

Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment

“…Thus, both hitherto assigned functional regions of pUL36 reside in the Nterminal part of the protein. It has been shown in a previous study that PrV particles devoid of the tegument proteins pUS3, pUL47 and pUL49 or pUS8 (the envelope glycoprotein E) incorporated the same amounts of several tegument proteins like pUL36, pUL37 and pUL47 as wild-type virions (Michael et al, 2006). This stoichiometric incorporation suggests that, like assembly of the capsid, the architecture of the tegument might, in part, be strictly controlled.…”

“…Mass lists were screened for peak pairs representing leucinecontaining pUL36-specific peptides in the conventional and mass-tagged variant and a mean intensity ratio, reflecting the relative amounts of protein originating from wild-type PrV or mutant virions, was calculated from all qualified peaks of one spectrum. Tryptic peptides derived from the major capsid protein, which is present in 960 copies (Steven & Spear, 1997) in every intact virion, were used as an internal standard (Michael et al, 2006). Values above 1?0 indicate an excess of the protein in the mutant particle.…”

“…On the other hand, amounts of other tegument proteins like pUL46, pUL48 and pUL49 varied to some extent among the different mutants, indicating also some degree of flexibility in tegument composition. The suggested interaction of pUL36 with pentonal sites on the capsid (Zhou et al, 1999), which are devoid of the small capsid protein pUL35 that decorates hexons, may partly explain this strict stoichiometry.In a study to analyse single-protein-deleted PrV mutants for alterations in the composition of the virion beyond the loss of the deleted protein, structural proteins in virus particles were quantified by a procedure designated 'stable isotope labelling by amino acids in cell culture' (SILAC) developed by Ong et al (2002), which was adapted for virions by our group (Michael et al, 2006). Virus particles from infected porcine kidney cell cultures were purified by sucrose-density-gradient centrifugation (Karger et al, 1998).…”

“…In a study to analyse single-protein-deleted PrV mutants for alterations in the composition of the virion beyond the loss of the deleted protein, structural proteins in virus particles were quantified by a procedure designated 'stable isotope labelling by amino acids in cell culture' (SILAC) developed by Ong et al (2002), which was adapted for virions by our group (Michael et al, 2006). Virus particles from infected porcine kidney cell cultures were purified by sucrose-density-gradient centrifugation (Karger et al, 1998).…”