Mutation of the nuclear localization signals of the major tegument protein pp65 leads to impairment of human cytomegalovirus progeny production

Abstract

The phosphoprotein 65 (pp65, pUL83) of human cytomegalovirus (HCMV) serves several functions during the viral life cycle. It is one of the most abundant constituents of the virion tegument and the major component of subviral particles termed Dense Bodies. It is also one of several tegument proteins that mediate the subversion of cellular antiviral defence functions like the induction of IFNs. It is also involved in the transcriptional activation of the major immediate early IE1/IE2 gene. Despite its strong nucleophilic nature, pp65 is found in the cytoplasm at late stages of infection. We have previously shown that the nuclear retention phenotype of a mutated pp65 is associated with a distinct deficit in viral productivity. In this study, we generated HCMV mutant viruses in which the nuclear localization signals (nls) of pp65 were mutated, thus preventing nuclear entry and pp65 shuttling. Since the genetic manipulation of UL83 led to the deregulation of the adjacent pp71 gene in initial experiments, we chose to express pp65 with mutated nls from a distant locus within the genome of a TB40/e strain derivative. This virus, termed v65stop-pp65nls, was significantly impaired in its capacity to produce infectious progeny. Using knockout fibroblast cells for the DNA sensors IFI16 or cGAS, no differences were seen when progeny production was compared between v65stop-pp65nls and a strain expressing wt pp65. Thus, differences in the known subversive effect on these DNA sensors could not account for the differences seen in productivity. Surprisingly, the upload of pp65 into virions and the synthesis of subviral Dense Bodies were abrogated by the mutation of its nls. Although an impairment of pp65 interaction with other proteins by the mutations cannot be excluded, the data suggest that the migration of pp65 through the nucleus was important for HCMV progeny production and packaging.