Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Abstract

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function. INTRODUCTIONHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that severely affects individuals with impaired or immature immune-defence functions (Griffiths et al., 2009;Mocarski et al., 2007). The components of the HCMV tegument have recently attracted considerable attention, as these proteins carry functions important for various stages of the virus replication cycle (reviewed by Kalejta, 2008). The phosphoprotein pp65 (ppUL83) has long been known as an abundant tegument constituent (Roby & Gibson, 1986), yet it is dispensable for HCMV growth in human foreskin fibroblast (HFF) cell cultures (Schmolke et al., 1995b).pp65 is an important target antigen of cellular and humoral immune responses (Beninga et al., 1995; McLaughlinTaylor et al., 1994; Plachter et al., 1990;Wills et al., 1996). It would thus be reasonable to assume that modification or deletion of the protein would be favourable for the virus. However, the sequence of pp65 is highly conserved in laboratory strains and clinical isolates (Dolan et al., 2004;Pande et al., 1991) and lack of pp65 expression in such strains has never been reported. Indeed, detection of pp65 has been used as a reliable diagnostic marker for acute HCMV infection in transplant recipients for over 15 years (Grefte et al., 1992a, b Initial studies described an association of pp65 with serine/ threonine kinase activity (Britt & Auger, 1986;Somogyi et al., 1990). Subsequent analyses identified the cellular Polo-like kinase 1 (Gallina et al., 1999) and the vi...

Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

“…In line with previous studies, phase-contrast images of cells infected with RV-VM1, but not with the wild-type parental strain RV-HB5, showed that NIBs (nuclear inclusion bodies) were undetectable in the majority of the infected nuclei and replaced by large globular structures (LGS) (Fig. 8A), both of which are considered sites of virus replication, transcription, and capsid assembly (33). Indirect immunofluorescence staining using a pp65 MAb demonstrated the nuclear retention of mutant pp65, especially at 144 hpi in cells infected with RV-VM1 (Fig.…”

“…The RV-HB5 virus was originally cloned by inserting a BAC vector into the US2-US6 gene region of the AD169 strain (35,36). The HCMV mutant RV-VM1, expressing nuclear pp65, is a descendant of RV-HB5 and was produced as previously reported (33). Briefly, pp65 in RV-VM1 carries a 30-amino-acid insertion at Arg387 that encompasses an immunodominant HLA-A2-presented peptide from the nonstructural IE1 protein (comprising amino acids 288 to 309) and a myc tag.…”

“…To gain more insight into the functional interaction between IFI16 and pp65 and establish whether this interaction is limited to MIEP activity modulation or could be extended to other viral gene promoters, we used a mutant of HCMV entirely lacking pp65 expression (v65Stop) (32) and a mutant unable to export pp65 from the nucleus (RV-VM1) (33). The results of our investigations demonstrate that pp65 is involved in the stabilization of IFI16 and in its nucleocytoplasmic dislocalization.…”

The Tegument Protein pp65 of Human Cytomegalovirus Acts as an Optional Scaffold Protein That Optimizes Protein Uploading into Viral Particles

“…RV-KB14 (28,32), which fail to produce subviral DBs; and additionally, two viral mutants, RV-SB2 and RV-VM1, in which pp65 was modified by insertion of a nonapeptide sequence at positions S101 and R387, respectively (33,34). DB formation is also abrogated in HFF infected with RV-SB2 and RV-VM1 (33,34). RV-VM1-infected HFF display wt pp65 steady-state protein levels (33), whereas RV-SB2 (a pp65low strain)-infected cells show a marked reduction in pp65 levels in relation to other virion proteins (Fig.…”

“…The pp65neg strain RV-KB14 was generated by inserting a tetracycline resistance cassette into the UL83 (pp65) ORF of pAD/cre (the BAC clone used for reconstitution of RV-BADwt), thereby deleting the pp65-coding region except for 152 5= base pairs of the ORF (32). The mutants RV-SB2 and RV-VM1 were generated by inserting the nonapeptide TMYGGISLL from the immediate-early 1 (IE1) protein of HCMV at position S101 or R387, respectively, of pp65, using galK-mediated recombination on the BACmid pHB5 (33,34). All viruses were characterized previously with respect to their genomic structures and biological properties.…”