Insertion of an EYFP-pp71 (UL82) coding sequence into the human cytomegalovirus genome results in a recombinant virus with enhanced viral growth.
The human cytomegalovirus (HCMV) UL82-encoded tegument protein pp71 has recently been shown to activate viral immediate-early (IE) gene expression by neutralizing a cellular intrinsic immune defense instituted by the ND10 protein hDaxx. Pp71 localizes to ND10 upon infection and induces the degradation of hDaxx. Here, we report the successful generation of a recombinant HCMV expressing enhanced yellow fluorescent protein (EYFP) fused to the N terminus of pp71. Intriguingly, insertion of the EYFP-UL82 coding sequence into the HCMV AD169 genome gave rise to a recombinant virus, termed AD169/EYFP-pp71, that replicates to significantly higher titers than wild-type AD169. In particular, we noticed strongly increased protein levels of pp71 after AD169/EYFP-pp71 inoculation. Although the high abundance of pp71 resulted in augmented packaging of the tegument protein into viral particles, no increased hDaxx degradation was detectable upon AD169/EYFP-pp71 infection. In contrast, further investigation revealed a significantly enhanced viral DNA replication compared to wild-type AD169. Thus, we hypothesize that an as-yet-unidentified function of pp71 contributes to the enhanced infectivity of AD169/EYFP-pp71. This assumption is additionally supported by the observation that increased early and late gene expression after AD169/EYFP-pp71 infection occurs independent of elevated IE protein levels. Finally, immunofluorescence analyses confirmed that hDaxx determines the ND10-localization of pp71 upon infection, since pp71 exhibited a nucleolar distribution in the absence of hDaxx. Taken together, we generated a recombinant HCMV that constitutes a useful tool not only to dissect the in vivo dynamics of pp71 subnuclear localization more precisely but also to explore new features of this viral transactivator.
Tavalai
Nina
N
Institute for Clinical and Molecular Virology, University Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany.
Kraiger
Martina
M
Kaiser
Nina
N
Stamminger
Thomas
T
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D002478:Cells, Cultured; D056970:Co-Repressor Proteins; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D018832:Molecular Chaperones; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*; D014779:Virus Replication
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D004279:DNA, Viral; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C054562:yellow fluorescent protein, Bacteria; C051298:cytomegalovirus phosphoprotein 71kDa