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Proteomic characterization of pseudorabies virus extracellular virions.

ABSTRACT: Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.

SUBMITTER: Kramer T

PROVIDER: S-EPMC3126529 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Proteomic characterization of pseudorabies virus extracellular virions.

Kramer T T Greco T M TM Enquist L W LW Cristea I M IM

Journal of virology 20110427 13

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virion ...[more]

PMID: 21525350

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Project description:The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-DeltaUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-DeltaUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.

Project description:The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-?NLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

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Proteomic characterization of pseudorabies virus extracellular virions.

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Proteomic characterization of pseudorabies virus extracellular virions.

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