Mutation of Glutamine to Arginine at Position 548 of IE2 86 in Human Cytomegalovirus Leads to Decreased Expression of IE2 40, IE2 60, UL83, and UL84 and Increased Transcription of US8-9 and US29-32

Abstract

The IE2 86 protein of human cytomegalovirus (HCMV) is essential for productive infection. The mutation of glutamine to arginine at position 548 of IE2 86 causes the virus to grow both slowly and to very low titers, making it difficult to study this mutant via infection. In this study, Q548R IE2 86 HCMV was produced on the complementing cell line 86F/40HA, which allowed faster and higher-titer production of mutant virus. The main defects observed in this mutant were greatly decreased expression of IE2 40, IE2 60, UL83, and UL84. Genome replication and the induction of cell cycle arrest were found to proceed at or near wild-type levels, and there was no defect in transitioning to early or late protein expression. Q548R IE2 86 was still able to interact with UL84. Furthermore, Q548R IE2 40 maintained the ability to enhance UL84 expression in a cotransfection assay. Microarray analysis of Q548R IE2 HCMV revealed that the US8, US9, and US29-32 transcripts were all significantly upregulated. These results further confirm the importance of IE2 in UL83 and UL84 expression as well as pointing to several previously unknown regions of the HCMV genome that may be regulated by IE2.

Analysis of the Functional Interchange between the IE1 and pp71 Proteins of Human Cytomegalovirus and ICP0 of Herpes Simplex Virus 1

“…Thus, the inducible cell system adds capabilities to the study that extend beyond what may be possible using viral mutants and certainly far beyond what is possible using transient expression methods. Inducible cell lines have also been used very effectively to study IE2 mutants (65).…”

The cytomegalovirus protein US31 induces inflammation through mono-macrophages in systemic lupus erythematosus by promoting NF-κB2 activation

“…However, the US31 gene identified during our analysis has not been investigated with regard to its expression mode and function. As expression of the proliferation-associated HCMV IE2 86 gene inhibits US31 expression, US31 may also be a HCMV latency-associated gene 27 . Notably, although neither Goodrum et al 18 or Cheung et al 26 detected the expression of US31, our data indicate that the positive rate of US31 gene expression in SLE patients is indeed higher compared to that in healthy control group, as confirmed using qPCR.…”

Crystal Structure of the DNA-Binding Domain of Human Herpesvirus 6A Immediate Early Protein 2

“…Fully conserved residues F1490 on ␣6, P1446 on ␣5, and C1344 on ␣1 constitute a hydrophobic cluster inside the fold. F1494 is the residue corresponding to Q548 of HCMV IE2, where substitution to arginine changed the IE2 activity (44), and resides on the adjacent region. The inset represents an overall view of IE2-CTD, and the red circle indicates the closeup region, although the observed angle was changed in the closeup view for clarity.…”

“…C-terminal ␣6 of HHV-6A IE2-CTD is another region that is absent from EBNA1-DBD and LANA-DBD. Helix ␣6 includes the residue corresponding to Q548 in HCMV IE2, for which replacement with arginine caused a virus replication defect and altered transcriptional activity (44). In the HHV-6A IE2-CTD structure, the side chains of the corresponding residue F1494 and the adjacent residue F1490 on ␣6 protrude toward the cavity surrounded by ␣3 of the HTH-like motif, N-terminal ␣1, and ␣5 and thus help to fix the spatial arrangement of these structural elements (Fig.…”