{'fraction_non_alphanumeric': 0.02481012658227848, 'fraction_numerical': 0.033417721518987344, 'mean_word_length': 5.5, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Insertion of an EYFP-pp71 (UL82) Coding Sequence into the Human Cytomegalovirus Genome Results in a Recombinant Virus with Enhanced Viral Growth

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.