Interactions of the KSHV nuclear egress complex: ORF69 is a potent factor for re-modeling cellular membranes.

Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Integrated Imaging Center.
Journal of Virology (Impact Factor: 4.44). 01/2013; 87(7). DOI: 10.1128/JVI.03418-12
Source: PubMed


All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC) that together facilitate the exit of assembled capsids from the nucleus. Previously we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by genes ORF67 and ORF69 when expressed in insect cells using baculoviruses for protein expression form a complex at the nuclear membrane and re-model these membranes to generate nuclear membrane derived vesicles. In this study we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and re-modeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C-terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67, however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type membrane anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation but all abolish re-modeling of nuclear membranes into circular structures. One mutant interacts with ORF67 as well as the wild-type protein but cannot function in membrane curvature and fission events that generate circular vesicles. These studies genetically confirm; that ORF67 is required for cellular membrane proliferation and ORF69 is the factor required to re-model these duplicated membranes into circular virion size vesicles. Furthermore, we also investigated the NEC encoded by Epstein-Barr virus (EBV). The EBV complex comprised of BFRF1 and BFLF2 was visualized at the nuclear membrane using autofluorescent protein fusions. BFRF1 is a potent inducer of membrane proliferation, however, BFLF2 cannot re-model these membranes into circular structures. What was evident is the superior re-modeling activity of ORF69, which could convert the host membrane proliferations induced by BFRF1 into circular structures.

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