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Insertion of an EYFP-pp71 (UL82) coding sequence into the human cytomegalovirus genome results in a recombinant virus with enhanced viral growth

 
: Tavalai, N.; Kraiger, M.; Kaiser, N.; Stramminger, T.

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Journal of virology 82 (2008), No.21, pp.10543-10555
ISSN: 0022-538X
ISSN: 1098-5514
English
Journal Article
Fraunhofer IOF ()
early gene-expression; herpes-simplex-virus; 58-KDA microspherule protein; intrinsic immune defense; open reading frame; tegument protein; infected cell; PP71 protein; intracellular-localization; mediated repression

Abstract
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.

: http://publica.fraunhofer.de/documents/N-84410.html