One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

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.

Membraneless Compartmentalization of Nuclear Assembly Sites during Murine Cytomegalovirus Infection

“…In contrast to pE1s, pIE1 remained scattered in the nucleus during 6–48 hpi, although pIE1 also concentrated within pE1-loaded spherical MLAs ( Figure 1 A). This is similar to the described behavior of IE1 and IE2 proteins in HCMV-infected cells [ 57 ] and IE3 protein in MCMV-infected cells [ 18 , 21 , 51 ].…”

“…Immediate-early 1 (pIE1, pm123) and early 1 (pE1, pM112-113) proteins are expressed very early after infection [ 18 , 19 , 21 , 22 , 27 , 28 , 49 , 50 , 51 , 52 ]. pIE1 is the product of the m123 gene, which belongs to the IE-class of regulatory proteins [ 49 ], while E1 proteins are the product of the M112-113 genes, which can generate at least four products of 33, 36, 38, and 87 kDa by alternative splicing [ 19 , 27 ], like their HCMV homologs UL112-113 [ 50 ].…”

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport

“…For the ORF3a mutagenesis, the plasmid pCAG-nCoV-ORF3a-HA that carries a WT ORF3a was used as a template to generate the respective ORF3a mutants as shown in Figure 2A using an overlapping PCR method ( Cruz-Cosme et al, 2020 ). Specifically, to generate the pCAG-nCoV-ORF3a-dDM2-HA plasmid that carries the domain two deletion (dDM2), two overlapping PCR fragments were made with two primer pairs: the primer pair 1: nCoV-ORF3a-HA-F and ORF3-HA-d36-40-up, and the primer pair 2: ORF3-HA-dDM2-down and nCoV-ORF3a-HA-R.…”