Nuclear pore composition and gating in herpes simplex virus-infected cells.
The mechanism by which herpes simplex virus (HSV) exits the nucleus remains a matter of controversy. The generally accepted route proposes that capsids exit via primary envelopment at the inner nuclear membrane and subsequent fusion of this primary particle with the outer nuclear membrane to gain capsid entry to the cytoplasm. However, recent observations indicate that HSV may induce gross morphological alterations of nuclear pores, resulting in the loss of normal pores and the appearance of dilated gaps in the nuclear membrane of up to several 100 nm. On this basis, it was proposed that a main route of capsid exit from the nucleus is directly through these altered pores. Here, we examine the biochemical composition of some of the major nuclear pore components in uninfected and HSV-infected cells. We show that total levels of major nucleoporins and their sedimentation patterns in density gradients remain largely unchanged up to 18 h after HSV infection. Some alteration in modification of one nucleoporin, Nup358/RanBP2, was observed during enrichment with anti-nucleoporin antibody and probing for O glycosylation. In addition, we examine functional gating within the nucleus in live cells, using microinjection of labeled dextran beads and a recombinant virus expressing GFP-VP16 to track the progress of infection. The nuclear permeability barrier for molecules bigger than 70 kDa remained intact throughout infection. Thus, in a functional assay in live cells, we find no evidence for gross perturbation to the gating of nuclear pores, although this might not exclude a small population of modified pores.
Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom.
Helmut
H
Hofemeister
Peter
P
O'Hare
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002213:Capsid / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008845:Microinjections; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry* / Q000502:physiology* / Q000821:virology; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D017405:Herpes Simplex Virus Protein Vmw65; D028861:Nuclear Pore Complex Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; C009434:tetramethylrhodamine isothiocyanate