Characterization of pseudorabies virus (PrV) cleavage-encapsidation proteins and functional complementation of PrV pUL32 by the homologous protein of herpes simplex virus type 1.
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.
Fuchs
Walter
W
Institute of Molecular Biology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Klupp
Barbara G
BG
Granzow
Harald
H
Leege
Tobias
T
Mettenleiter
Thomas C
TC
D000818:Animals; D002213:Capsid / Q000648:ultrastructure; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D022202:Chromosomes, Artificial, Bacterial; D003593:Cytoplasm / Q000737:chemistry; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D011558:Herpesvirus 1, Suid / Q000502:physiology*; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; C069185:DNA cleavage and packaging proteins, Herpesvirus