Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Abstract

Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5=-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5= stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5= deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5= deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7-and 21-nucleotide deletions. Since 5= stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CD pro , to deleted positive-strand RNAs corresponding to the 5= end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CD pro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5=-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5= end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5=-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis as-

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets

“…Interestingly, a series of recent studies has shown that persistent coxsackievirus genomes mutate, undergoing 5=-terminal deletions (ϳ7 to 49 nucleotides [nt]) that are associated with long-term viral persistence (43, 45-48). Notably, the 5=-terminally deleted picornavirus genomes have been shown to be capable of replicating, resulting in low-yield infectious virus production (48,49). Collectively, these observations suggest at least two potential mechanisms for picornavirus genome persistence: (i) continuous low-level replication in persistently infected cells and/or (ii) cycles of productive and nonproductive infection.…”

“…Swabs (oral, nasal, and rectal/fecal) and blood samples (serum and whole heparinized blood) were collected during the acute phase (days 0, 1, 3, 5, 7, 14, 21, 28, and 35 p.i.) and chronic/persistent phase (days 46,47,48,49,50,51,52,53,54,55,56,57,58,59, and 60 p.i.) of the experiment.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…This oriL-3CD interaction involves the hairpin domain d of oriL and the 3C pro moiety of 3CD, as originally demonstrated by Andino et al (90,94). The interaction between these ligands is important for the initiation of the synthesis of both the viral (positive) and complementary (negative) RNA strands (89,93,(95)(96)(97)(98). It was recently shown that (at least in the case of coxsackievirus B3 [CVB3]) oriL, which is required for efficient genome replication, is not indispensable for viral viability: its removal does not kill the virus but rather decreases the efficiency of its RNA replication ϳ10 5 -fold (99).…”

“…2A). This element plays multiple roles in viral reproduction, mainly through promoting formation of a complex ribonucleoprotein (RNP) structure involving several viral and host proteins (89)(90)(91)(92)(93). An essential component of this complex is the viral RdRP (3D pol ), which is recruited there in the form of its precursor, 3CD, i.e., covalently linked to the viral protease (3C pro ) (91).…”

Immunological and pathological consequences of coxsackievirus RNA persistence in the heart

“…That these deletions would impede viral RNA replication is consonant with the fact that the first ~100 bases of the 5′ noncoding region of CVB3 (and other enteroviruses) are folded into a structure that visually resembles a clover leaf, and plays a vital role in the syntheses of both positive and negative strands. Some time ago, our laboratory reported that deletion of as few as 32 bases at the 5′ end of the CVB3 cloverleaf had a profound negative impact on CVB3 replication, but had minimal impact on the molecule’s suitability as a template for translation (Hunziker et al, 2007), and more recent studies have shown that TD virus replication is ~10 5 -fold less robust than that of the wt virus (Jaramillo et al, 2016; Leveque et al, 2017; Smithee et al, 2015). TD CVBs also have been identified in humans.…”