Abstract
The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.
Highlights
Murine cytomegalovirus (MCMV) infection in mice is the most often used small animal model for investigating human CMV (HCMV) pathogenesis due to the similarities between Human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) [1].CMV belongs to the beta-subfamily of the Herpesviridae family, and it contains an about-235-kb DNA genome that encodes about 200 genes [2]
In a transfection system using M112-113-expressing plasmid, where the M112-113 gene is under the control of its own promoter, we found that M112-113 proteins can be expressed at a low level without immediate-early 3 (IE3), but IE3 enhanced the M112-113 gene expression to a much higher level [9]
We found that one of the triple poly(A) signals is enough for M112-113 gene expression, and the PASs are important for MCMV replication
Summary
CMV belongs to the beta-subfamily of the Herpesviridae family, and it contains an about-235-kb DNA genome that encodes about 200 genes [2]. CMV gene expression is sequentially regulated in the infected cells, and CMV genes are defined as immediate-early (IE), early (E), and late (L). IE gene expression is independent of any viral de novo gene expression; E gene expression depends on IE proteins, and early proteins are important for viral DNA replication and late gene expression [2]. The mechanism of how IE proteins regulate E gene expression has been a focus of studies in the CMV field, and it is still elusive. The M112-113 gene is thought to be the first gene expressed at the early stage (i.e., after the immediate early stage) after MCMV infection in permissive cells, so it is called E1
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