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.

Fig. 1.

RhCMV and MCMV express RNAs from the equivalent genomic locus as that encoding the HCMV 5-kb intron. (A) The 5-kb RNA locus of HCMV. Gray arrows indicate previously annotated ORFs that are unlikely to encode proteins. (B) (Upper) The RhCMV UL112/113-UL105 locus. Locations of probes A–D are indicated. (Lower Left) Northern blot analysis of total RNA prepared from mock-infected (M) or RhCMV-infected rhesus fibroblasts at 48 h postinfection (hpi) with probes indicated below the blot. (Lower Right) Kinetics of RhCMV 3-kb RNA expression were analyzed by Northern blot analysis of total RNA prepared at the indicated times after infection by hybridization with a combination of probes C and D. (C) (Upper) The MCMV UL112/113–UL105 locus. Gray arrows indicate previously annotated ORFs that are unique to the MCMV genome (m106, m107, and m108) and not known to encode proteins. Locations of probes F and G are indicated. (Lower) Northern blot analysis of total RNA prepared from mock-infected (M) or MCMV-infected mouse fibroblasts at the indicated times after infection with the probes indicated below the blots. Cross-hybridization to the 28S rRNA (lower band) often is detected and is an artifact of the use of riboprobes for hybridization.

Fig. 2.

Identification of 7.2- and 8.0-kb introns encoded by the MCMV genome. (A) Mapping the 5′ end of the 7.2-kb RNA by primer extension analysis. Total RNA was prepared from mock-infected (M, lane 2) or MCMV-infected (24 h, lane 3) mouse fibroblasts. Lane 1 contains a radiolabeled 10-bp ladder, and lane 4 contains a radiolabeled 88-bp control oligonucleotide. (B) Identification of splice junctions by RT-PCR analysis of the 7.2- and 8.0-kb RNA locus. RT-PCRs were performed with primers 69 and 106.5 (Left) or 28 and 106.5f (Right) by using total RNA prepared from mock-infected or MCMV-infected fibroblasts at 24 and 48 h after infection. RT-PCR products were analyzed by agarose gel electrophoresis. (C) The MCMV 7.2-kb RNA locus. The genomic coordinates of the 7.2-kb and 8.0-kb introns as well as three MCMV-specific ORFs are indicated, and the locations of primers used in the primer extension and RT-PCR assays are shown.

Fig. 3.

The locus encoding the 7.2- and 8.0-kb introns is required for progression of MCMV infection to the persistent phase. (A) Structure of the intron locus-deficient, dl7.2 + 8.0kb virus. The locations of the 7.2- and 8.0-kb introns are displayed, and a thick bar (sequence position 162,078 to 154,056) indicates the genomic sequence deleted and replaced with Kan/LacZ cassette in dl7.2 + 8.0. (B) Northern blot hybridization with probe G of total RNA from fibroblasts harvested at 24 h after mock infection or infection with wild-type (WT), mutant (dl7.2 + 8.0kb), or revertant (rev7.2 + 8.0kb) virus. (C) In vitro analysis of recombinant virus replication. Fibroblasts were infected at a multiplicity of 0.05 pfu/cell with indicated viruses. Culture supernatants were collected daily and titered by plaque assay on fibroblasts. (D) In vivo analysis of recombinant virus replication. Six-week-old female BALB/c mice were infected with an i.p. dose of 1 × 10⁶ pfu with indicated viruses. Total yield of infectious virus per organ is reported for the indicated days postinfection (dpi), as measured by plaque assay of organ homogenates on mouse fibroblasts.

Fig. 4.

The 7.2-kb intron is required for progression of MCMV infection to the persistent phase. (A) Structure of recombinant viruses in the MCMV 7.2-kb RNA locus. The locations of the 7.2- and 8.0-kb introns are displayed, and the genomic coordinates are indicated for alterations in the pmSD1 splice-donor substitution mutant (GGTAG to CTAGT) that blocks production of the 7.2-kb intron, the sub3.1 substitution mutant (deletion of genomic sequence and insertion of Kan/LacZ marker cassette) that encodes an enlarged 7.2-kb intron, and the subHP1 hairpin motif substitution mutant (59-bp deletion plus an insertion of a single FRT site, a sequence required for action of the yeast flp recombinase) that destabilizes the 7.2-kb intron. (B) Northern blot hybridization with a combination of probes G and p640 of total RNA harvested at 24 h after mock infection or infection with the indicated viruses. (C) In vitro analysis of recombinant virus replication. Fibroblasts were infected with the indicated viruses at a multiplicity of 0.05 pfu/cell. Culture supernatants were collected daily and titered by plaque assay on fibroblasts. (D) In vivo analysis of recombinant virus replication. Six-week-old, female BALB/c mice were infected with an i.p. dose of 1 × 10⁶ pfu with indicated viruses. Total yield of infectious virus per organ is reported for the indicated days postinfection (dpi), as measured by plaque assay of organ homogenates on mouse fibroblasts.