Abstract
Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods: We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results: CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions: Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.
Highlights
Capsids of herpes simplex virus 1 (HSV-1) are assembled in host cell nuclei, transported to the nuclear periphery and translocated to the perinuclear space (PNS) or into the cytosol (Roizman et al, 2007)
CK177ΔUs3gK-HA induces alteration of nuclear and Golgi membranes Infection with HSV-1ΔUs3 induces formation of folds and invagination of nuclear membranes associated with accumulation of virions (Poon et al, 2006; Reynolds et al, 2002; Wisner et al, 2009)
Homogenous, proteinaceous structures occurred within nuclei and cytoplasm of CK177ΔUs3gK-HA (Figure 1) and R7041ΔUs3 (Figure 2A) infected cells
Summary
Capsids of herpes simplex virus 1 (HSV-1) are assembled in host cell nuclei, transported to the nuclear periphery and translocated to the perinuclear space (PNS) or into the cytosol (Roizman et al, 2007). Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. Results: CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity
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