Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse

Abstract

The human cytomegalovirus immediate-early 5-kb RNA previously has been shown to be a stable intron that is not required for efficient replication of the virus in cultured fibroblasts. Here we describe a murine cytomegalovirus 7.2-kb ortholog of the human cytomegalovirus 5-kb RNA. The 5 end of the 7.2-kb transcript maps to a consensus splice-donor site that is conserved among all cytomegaloviruses. We constructed mutant viruses lacking the entire 7.2-kb coding domain, the splice-donor site predicted to function in the generation of the intron or a hairpin predicted to stabilize the intron. All three mutant viruses failed to produce the 7.2-kb RNA, supporting our conclusion that it is a stable intron. Each of the mutants replicated with normal kinetics in cultured fibroblasts, but the mutants exhibited a clear defect within infected mice. Although the initial acute phase at 4 days after infection appeared to be normal, none of the mutant viruses progressed to the persistent phase, i.e., little virus was detected in the salivary gland at 14 days after infection. The intron functions as an in vivo virulence factor, facilitating progression from the acute to persistent phase of infection.RNA processing ͉ salivary gland ͉ viral pathogenesis H uman cytomegalovirus (HCMV) is a member of the ␤-herpesvirus family and a widespread human pathogen (1). Primary HCMV infection is generally asymptomatic in healthy individuals, and, after limited acute replication, virus replication persists in epithelial cells of the salivary gland, mammary gland, and kidney. These sites of persistence allow the virus to spread to new hosts via saliva, breast milk, and urine (2).During productive infection, HCMV genes are expressed in an organized cascade of transcription (1). Immediate-early genes are expressed first and encode proteins that establish a cellular environment conducive to viral replication. One immediateearly transcript, known as the 5-kb RNA (3-6), is not known to encode a protein. We have demonstrated that this RNA is an intron that accumulates in the nuclei of infected cells (5).The genomic sequence from which the 5-kb RNA is expressed resides between the UL105 and UL112͞113 loci (Fig. 1A) and has an AT content of Ϸ60%, considerably higher than that of the overall HCMV genome. Six ORFs (UL106-UL111), ranging in length from 93 to 153 aa, have been annotated in the region spanned by the 5-kb RNA, and numerous stop and start codons define additional small ORFs (7). Although the DNA sequences of the 5-kb loci from laboratory-adapted and clinical isolates of HCMV are well conserved, the predicted ORFs vary considerably because of single-nucleotide changes, insertions, and deletions (8, 9). There is no compelling evidence that any one of these ORFs encodes a protein. A polyadenylated 1.1-kb RNA (10) is generated by splicing the 5-kb intron from its primary transcript (5), and it is predicted to encode a 31-aa polypeptide that bears no similarity to other known proteins. A variant of the AD169 strain of HCMV unable to produce the 5-...

Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes

“…The cytoplasmic LAT exists in a nonlinear form, presumably a lariat (Wu et al 1996;Rodahl and Haarr 1997). A similar stable intron is derived from the murine cytomegalovirus (Kulesza and Shenk 2006). Finally, in a recent study it was shown that lariat introns accumulate in the cytoplasm of dbr1Δ yeast cells (Armakola et al 2012).…”

T-box transcription factor T-bet, a key player in a unique type of B-cell activation essential for effective viral clearance

“…gHV68 virus was generated as was previously described (49). MCMV was obtained from Dr. C. Kulesza (Princeton University, Princeton) and was generated as previously described (50). LCMV was obtained from Dr. D. Homann (University of Colorado, Denver) and was generated as previously described (51).…”

Discrete Clusters of Virus-Encoded MicroRNAs Are Associated with Complementary Strands of the Genome and the 7.2-Kilobase Stable Intron in Murine Cytomegalovirus

“…A recent report by Kulesza and Shenk (21) demonstrated that MCMV encodes a 7.2-kb stable intron that occurs in this vicinity; a less abundant 8.0-kb intron was also reported (21), which would encompass the miRNAs in this cluster (Fig. 3A, inset).…”