5′-Terminal Deletions Occur in Coxsackievirus B3 during Replication in Murine Hearts and Cardiac Myocyte Cultures and Correlate with Encapsidation of Negative-Strand Viral RNA

Abstract

Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5 termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5 CG or 5 AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.The six serotypes of the group B coxsackieviruses (CVB1-6) are enteroviruses (Picornaviridae, species HEV-B) (53). The CVB genome is a single-stranded RNA molecule, 7,400 nucleotides (nt) in length, that is encapsidated within an icosahedral shell (74). The 11 viral proteins (83) are encoded by a single open reading frame which is flanked on the 5Ј and 3Ј termini by nontranslated regions (NTRs) (20). The CVB induce numerous human illnesses, including inflammatory heart disease, pancreatitis, and aseptic meningitis and may also trigger the onset of type 1 diabetes (5,31,72,81,82). The CVB were recognized as causes of human heart disease shortly after their description early in the 1950s (26, 27) and remain the enteroviruses most commonly associated with human cardiomyopathies on the grounds of isolation and serology (5,6,34,62,88). Meta-analysis shows an association between enteroviruses and myocarditis in 23% of cases (7), although this association is more variable in cases of dilated cardiomyopathy, a serious disease that often leads to a failing heart (61). Mouse models of CVB-induced pancreatitis (94, 110), myocarditis (45,52,110), myositis (104, 105), and rapid-onset type 1 diabetes (31) which facilitate the study of these diseases have been developed.Enterovirus infections are generally considered to be acute events, with symptoms and virus titers peaking within a few days postinoculation (p.i.) and with virus being cleared by the adaptive immune response (17). However, enterovirus infections can persist under conditions of immunodeficiency (48,51,...

How RNA viruses maintain their genome integrity

“…In the case of CB3V, terminal deletions arise more rapidly in either intact heart tissues or primary myocardial cell cultures than in other cells, suggesting that the cellular environment has a strong influence (Kim et al, 2005). However, one virus associated with persistent virus infections appears to have evolved a specific strategy for truncating its termini.…”

“…CB3V genomes can be detected in tissue from previously infected individuals, yet infectious viruses have been almost impossible to isolate, suggesting that CB3V may be capable of viral persistence. Evidence for a persistence mechanism came from sequence analysis, which showed that CB3V genomes within infected tissues possessed 59-terminal deletions of between 7 and 49 nt (Kim et al, 2005). It was found that there was a correlation between the presence of complete or truncated termini, and acute versus persistent infection, respectively (Kim et al, 2005).…”

“…Evidence for a persistence mechanism came from sequence analysis, which showed that CB3V genomes within infected tissues possessed 59-terminal deletions of between 7 and 49 nt (Kim et al, 2005). It was found that there was a correlation between the presence of complete or truncated termini, and acute versus persistent infection, respectively (Kim et al, 2005). More recent work has detected the presence of terminally deleted genomes in human tissue collected from a fatal case of myocarditis, suggesting that there is significance for terminal deletions in the context of a human infection (Chapman et al, 2008).…”

Replication of Coxsackievirus B3 in Primary Cell Cultures Generates Novel Viral Genome Deletions

“…Human enteroviruses cause acute myocarditis and are etiologically linked as causes of dilated cardiomyopathy, a disease that can proceed to heart failure (2,3,11,20,22). Enteroviral RNA persistence in adult hearts with myocarditis or dilated cardiomyopathy in the absence of detectable infectious virus has been a common observation (reviewed in references 3, 11, and 12) but remained without adequate explanation until we demonstrated that, during replication in isolated murine cardiomyocytes and in hearts of infected mice, CVB3 can naturally delete sequence information at the genomic 5Ј terminus (13). This work showed that, weeks following the inoculation of mice with CVB3, mouse myocardium in which no cytolytic virus was evident was able to transmit virus to cell cultures, which, in turn, showed no cytopathic effects (CPE) despite the detection of CVB3 RNA.…”

“…In addition, purified CVB3TD virions encapsidate both positive-and negative-strand RNA in a strand ratio similar to that of total RNA from CVB3TD-infected cell cultures. Because CPE in CVB3TD-infected cell cultures are not measurable, a method was developed to titer infectious CVB3TD using real-time quantitative reverse transcription-PCR (RT-PCR) analysis of the viral RNA content in CVB3TD preparations (13). It was not surprising to note deviations from wild-type enteroviral replication, as the region in the 5Ј NTR that is progressively deleted (the cloverleaf RNA structure termed domain I) is also an essential region for viral replication (1,17,19).…”