Herpes simplex virus tegument ICP0 is capsid associated, and its E3 ubiquitin ligase domain is important for incorporation into virions

Abstract

Herpes simplex virus (HSV) immediate-early (IE) protein ICP0 is a multifunctional regulator of HSV infection. ICP0 that is present in the tegument layer has not been well characterized. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for IE functions. Virions with mutations in this domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins.

FIG. 1.

Protein composition of herpes simplex virions in the absence of ICP0. (A) The HSV-1 wild type (17⁺) or ΔICP0 strain (dl1403) was analyzed by SDS-PAGE followed by Coomassie blue staining. One microgram (left) or equivalent VP5 units (right) of each virus were loaded. In the center, locations of several HSV structural proteins are indicated. Numbers to the left are molecular mass markers in kilodaltons. (B) Equivalent VP5 units of extracellular virions were analyzed by SDS-PAGE followed by Western blotting with antibodies against the indicated tegument protein or glycoprotein.

FIG. 2.

Effect of RING finger mutations on the incorporation of ICP0 into virions. (A) Incorporation of tegument ICP0 into HSV-1 mutants. The indicated extracellular virions (identified by relevant mutations where applicable) were analyzed by SDS-PAGE followed by Western blotting with the 11060 antibody to ICP0. In parallel, VP5 was detected by Coomassie staining to demonstrate equivalent levels of particle loading. dl1403R and FXER, dl1403 and FXE rescuants carrying a restored ICP0 gene. (B) Expression of ICP0 in cells infected with wild-type or mutant viruses. U2OS cells were infected with the indicated virus (MOI = 1) for 18 h. Cell lysates were analyzed by SDS-PAGE followed by Western blotting with MAb H1A027 to ICP0.

FIG. 3.

Sensitivity of tegument ICP0 to detergent lysis and salt treatment. Extracellular virions were treated with 1% Triton X-100 in the presence of 0.1, 0.5, or 1 M NaCl. The reaction mixtures were then centrifuged through a 35% sucrose cushion. Pelleted (pellet) and released (sup) fractions were analyzed by SDS-PAGE and immunoblotting with antibodies to ICP0, VP1-2, ICP4, VP16, gB, or VP5.