Functional Analysis of Human Cytomegalovirus (HCMV) US3 and pp71
Abstract (Summary)Human cytomegalovirus (HCMV), a betaherpesvirus, is an opportunistic pathogen and causes serious diseases in immunocompromised individuals and the developing fetuses. HCMV utilizes a number of mechanisms to evade detection by the immune system. US3, an immediate early gene of HCMV, is expressed shortly after infection and plays an important role in immune evasion by inhibiting the maturation of MHC class I heavy chains. The US3 gene is transcribed into three alternatively spliced RNAs that encode distinct but related peptides. The unspliced US3 RNA encodes a 19-kDa ER resident protein that consists of a lumenal domain, a transmembrane domain and a short cytoplasmic tail. A 17kDa-protein encoded by a singly-spliced US3 RNA has 134 amino acids in common with the 19 kDa protein but is unable to retain MHC class I heavy chains in the ER. The 17-kDa protein does not contain a transmembrane domain, suggesting that the transmembrane domain of the 19-kDa protein is critical for the immune evasion mechanism of the US3 gene. Deletion analyses of the 19 kDa protein confirmed the functional significance of the transmembrane domain as well as its significance in determining the intracellular location of the protein. Site-directed mutagenesis has demonstrated that Cys44 and Cys129 form an intracellular disulfide bond critical for the function of the 19-kDa US3 protein. The significance of the carboxyl terminal KK motif was also investigated. pp71, encoded by the UL82 gene, is a potent activator of the mIE transcription immediately after infection via enhancer sequences containing the ATF and AP-1 binding sites. It can also increase the infectivity of viral genomic DNA dramatically through an unknown mechanism. pp71 has no homology or similarity to other known proteins. A series of amino acid deletions or point mutations of the protein were expressed and examined for their effects on transactivation of the mIE promoter and infectivity of viral genomic DNA. Short truncations at the N-terminus had slight effects while longer truncations at both termini and internal deletions dramatically impaired the transactivation ability of pp71. The C-terminal acidic amino acid stretch, which serves as a functional domain in many viral transactivators, is not essential for its function. All domains contribute to the integral function of pp71 and are sensitive to perturbation for both transactivation and infectivity of genomic DNA.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:hcmv us3 mhc class 1 pp71
Date of Publication:01/01/2001