Nuclear import of the varicella-zoster virus latency-associated protein ORF63 in primary neurons requires expression of the lytic protein ORF61 and occurs in a proteasome-dependent manner.
Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shown to be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Matthew S
MS
Walters
Christos A
CA
Kyratsous
Shilin
S
Wan
Saul
S
Silverstein
D021581:Active Transport, Cell Nucleus; D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D017615:Enteric Nervous System / Q000166:cytology; D004847:Epithelial Cells / Q000821:virology; D019084:Fluorescent Antibody Technique, Indirect; D005724:Ganglia / Q000166:cytology; D005982:Glutathione Transferase / Q000378:metabolism; D006168:Guinea Pigs; D006562:Herpes Zoster / Q000821:virology; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000821:virology*; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000821:virology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D017735:Virus Latency
D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C074551:immediate early protein 63, Human herpesvirus 3; C073411:protein 61, Varicella-zoster virus; D049410:Luciferases, Renilla; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex