Intracellular localization of the pseudorabies virus large tegument protein pUL36.
Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated "inner" tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.
Möhl
Britta S
BS
Institutes of Molecular Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.
Böttcher
Sindy
S
Granzow
Harald
H
Kuhn
Jana
J
Klupp
Barbara G
BG
Mettenleiter
Thomas C
TC
D020816:Amino Acid Motifs; D000818:Animals; D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D011558:Herpesvirus 1, Suid / Q000378:metabolism*; D007668:Kidney / Q000821:virology; D008856:Microscopy, Fluorescence / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014764:Viral Proteins / Q000096:biosynthesis / Q000502:physiology*; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000502:physiology*
D017931:DNA Primers; D014764:Viral Proteins; D015678:Viral Structural Proteins; D049452:Green Fluorescent Proteins