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Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins.

ABSTRACT: Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.

SUBMITTER: Kuhn J

PROVIDER: S-EPMC2395125 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins.

Kuhn Jana J Leege Tobias T Klupp Barbara G BG Granzow Harald H Fuchs Walter W Mettenleiter Thomas C TC

Journal of virology 20080409 12

Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear e ...[more]

PMID: 18400859

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Project description:Cleavage and encapsidation of newly replicated herpes simplex virus type 1 (HSV-1) DNA requires several essential viral gene products that are conserved in sequence within the Herpesviridae. However, conservation of function has not been analyzed in greater detail. For functional characterization of the UL6, UL15, UL28, UL32, and UL33 gene products of pseudorabies virus (PrV), the respective deletion mutants were generated by mutagenesis of the virus genome cloned as a bacterial artificial chromosome (BAC) in Escherichia coli and propagated in transgenic rabbit kidney cells lines expressing the deleted genes. Neither of the PrV mutants was able to produce plaques or infectious progeny in noncomplementing cells. DNA analyses revealed that the viral genomes were replicated but not cleaved into monomers. By electron microscopy, only scaffold-containing immature but not DNA-containing mature capsids were detected in the nuclei of noncomplementing cells infected with either of the mutants. Remarkably, primary envelopment of empty capsids at the nuclear membrane occasionally occurred, and enveloped tegument-containing light particles were formed in the cytoplasm and released into the extracellular space. Immunofluorescence analyses with monospecific antisera of cells transfected with the respective expression plasmids indicated that pUL6, pUL15, and pUL32 were able to enter the nucleus. In contrast, pUL28 and pUL33 were predominantly found in the cytoplasm. Only pUL6 could be unequivocally identified and localized in PrV-infected cells and in purified virions, whereas the low abundance or immunogenicity of the other proteins hampered similar studies. Yeast two-hybrid analyses revealed physical interactions between the PrV pUL15, pUL28, and pUL33 proteins, indicating that, as in HSV-1, a tripartite protein complex might catalyze cleavage and encapsidation of viral DNA. Whereas the pUL6 protein is supposed to form the portal for DNA entry into the capsid, the precise role of the UL32 gene product during this process remains to be elucidated. Interestingly, the defect of UL32-negative PrV could be completely corrected in trans by the homologous protein of HSV-1, demonstrating similar functions. However, trans-complementation of UL32-negative HSV-1 by the PrV protein was not observed.

Project description:Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

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Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins.

Publications

Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins.

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