Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

A novel population of myeloid cells responding to coxsackievirus infection assists in the dissemination of virus within the neonatal CNS.

Enterovirus infection in newborn infants is a significant cause of aseptic meningitis and encephalitis. Using a neonatal mouse model, we previously determined that coxsackievirus B3 (CVB3) preferentially targets proliferating neural stem cells located in the subventricular zone within 24 h after infection. At later time points, immature neuroblasts, and eventually mature neurons, were infected as determined by expression of high levels of viral protein. Here, we show that blood-derived Mac3(+) mononuclear cells were rapidly recruited to the CNS within 12 h after intracranial infection with CVB3. These cells displayed a myeloid-like morphology, were of a peripheral origin based on green fluorescent protein (GFP)-tagged adoptive cell transplant examination, and were highly susceptible to CVB3 infection during their migration into the CNS. Serial immunofluorescence images suggested that the myeloid cells enter the CNS via the choroid plexus, and that they may be infected during their extravasation and passage through the choroid plexus epithelium; these infected myeloid cells ultimately penetrate into the parenchyma of the brain. Before their migration through the ependymal cell layer, a subset of these infected myeloid cells expressed detectable levels of nestin, a marker for neural stem and progenitor cells. As these nestin(+) myeloid cells infected with CVB3 migrated through the ependymal cell layer, they revealed distinct morphological characteristics typical of type B neural stem cells. The recruitment of these novel myeloid cells may be specifically set in motion by the induction of a unique chemokine profile in the CNS induced very early after CVB3 infection, which includes upregulation of CCL12. We propose that intracranial CVB3 infection may lead to the recruitment of nestin(+) myeloid cells into the CNS which might represent an intrinsic host CNS repair response. In turn, the proliferative and metabolic status of recruited myeloid cells may render them attractive targets for CVB3 infection. Moreover, the migratory ability of these myeloid cells may point to a productive method of virus dissemination within the CNS.

Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.

Coxsackieviruses are significant human pathogens, and the neonatal central nervous system (CNS) is a major target for infection. Despite the extreme susceptibility of newborn infants to coxsackievirus infection and viral tropism for the CNS, few studies have been aimed at determining the long-term consequences of infection on the developing CNS. We previously described a neonatal mouse model of coxsackievirus B3 (CVB3) infection and determined that proliferating stem cells in the CNS were preferentially targeted. Here, we describe later stages of infection, the ensuing inflammatory response, and subsequent lesions which remain in the adult CNS of surviving animals. High levels of type I interferons and chemokines (in particular MCP-5, IP10, and RANTES) were upregulated following infection and remained at high levels up to day 10 postinfection (p.i). Chronic inflammation and lesions were observed in the hippocampus and cortex of surviving mice for up to 9 months p.i. CVB3 RNA was detected in the CNS up to 3 months p.i at high abundance ( approximately 10(6) genomes/mouse brain), and viral genomic material remained detectable in culture after two rounds of in vitro passage. These data suggest that CVB3 may persist in the CNS as a low-level, noncytolytic infection, causing ongoing inflammatory lesions. Thus, the effects of a relatively common infection during the neonatal period may be long lasting, and the prognosis for newborn infants recovering from acute infection should be reexplored.

Coxsackievirus B3 proteins directionally complement each other to downregulate surface major histocompatibility complex class I.

Picornaviruses carry a small number of proteins with diverse functions that subvert and exploit the host cell. We have previously shown that three coxsackievirus B3 (CVB3) proteins (2B, 2BC, and 3A) target the Golgi complex and inhibit protein transit. Here we investigate these effects in more detail and evaluate the distribution of major histocompatibility complex (MHC) class I molecules, which are critical mediators of the CD8(+) T-cell response. We report that concomitant with viral protein synthesis, MHC class I surface expression is rapidly downregulated during infection. However, this phenomenon may not result solely from inhibition of anterograde trafficking; we propose a new mechanism whereby the CVB3 2B and 2BC proteins upregulate the internalization of MHC class I (and possibly other surface proteins), perhaps by focusing of endocytic vesicles at the Golgi complex. Thus, our findings indicate that CVB3 carries at least three nonstructural proteins that directionally complement one another; 3A disrupts the Golgi complex to inhibit anterograde transport, while 2B and/or 2BC upregulates endocytosis, rapidly removing proteins from the cell surface. Taken together, these effects may render CVB3-infected cells invisible to CD8(+) T cells and untouchable by many antiviral effector molecules. This has important implications for immune evasion by CVB3.

Deletions within the 5'UTR of coxsackievirus B3: consequences for virus translation and replication.

Key features of an ideal RNA-based vaccine against coxsackievirus B3 (CVB3) are (i) limited genome replication/virus production (to minimize vaccine-related pathology) and (ii) abundant virus protein synthesis (to maximize immunogenicity). These attributes may apply to CVB3 RNAs lacking up to 250 nucleotides (nt) from their 5' terminus; these RNAs do not give rise to infectious progeny, but they have been reported to retain the entire CVB3 IRES (mapped to nt approximately 432-639) and to produce large quantities of viral protein in transfected cells. Here, we constructed five 5' RNA deletion variants that, to our surprise, failed to protect against CVB3 challenge. We investigated the reasons for this failure and conclude that (i) a 5' terminal deletion as short as 32 nt abolishes CVB3 RNA replication in transfected cells; (ii) this deleted RNA, and others with longer deletions, do not direct abundant protein synthesis in transfected cells, probably as a consequence of their replicative incapacity; and (iii) the CVB3 IRES is substantially larger than previously thought, and its 5' boundary lies between residues 76 and 125, very closely approximating that of the poliovirus IRES.

Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle.

Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.

Host and virus determinants of picornavirus pathogenesis and tropism.

The family Picornaviridae contains some notable members, including rhinovirus, which infects humans more frequently than any other virus; poliovirus, which has paralysed or killed millions over the years; and foot-and-mouth-disease virus, which led to the creation of dedicated institutes throughout the world. Despite their profound impact on human and animal health, the factors that regulate pathogenesis and tissue tropism are poorly understood. In this article, we review the clinical and economic challenges that these agents pose, summarize current knowledge of host-pathogen interactions and highlight a few of the many outstanding questions that remain to be answered.

An authentic 3' noncoding region is necessary for efficient poliovirus replication.

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

Analysis of translational initiation in coxsackievirus B3 suggests an alternative explanation for the high frequency of R+4 in the eukaryotic consensus motif.

Translational initiation of most eukaryotic mRNAs occurs when a preinitiation complex binds to the 5' cap, scans the mRNA, and selects a particular AUG codon as the initiation site. Selection of the correct initiation codon relies, in part, on its flanking residues; in mammalian cells, the core of the "Kozak" consensus is R-3CCAUGG+4 (R=purine; the A residue is designated position +1). The R-3 is considered the most important flanking residue, followed by G+4. Picornaviral mRNAs differ from most cellular mRNAs in several ways; they are uncapped, and they contain an internal ribosome entry site that allows the ribosome to bind near the initiation codon. The initiation codon of coxsackievirus B3 (CVB3) is flanked by both R-3 and G+4 (AAAATGG). Here, we report the construction of full-length CVB3 genomes that vary at these two positions, and we evaluate the effects of these variant sequences in vitro, in tissue culture cells, and in vivo. A virus with an A-->C transversion at position -3 replicates as well as wild-type CVB3, both in tissue culture and in vivo. This virus is highly pathogenic, and its sequence is stable throughout the course of an in vivo infection. Furthermore, the in vitro translation products from this RNA are very similar to the wild type. Thus, R-3-thought to be the most functionally important component of the Kozak consensus-appears to be dispensable in CVB3. In contrast, a G-to-C transversion at G+4 is lethal; RNAs carrying this mutation fail to generate infectious virus either in tissue culture or in vivo. However, in vitro analysis indicates that G+4 has only a marginal effect on translational initiation, especially if R-3 is present; instead, the G+4 is required mainly because the second triplet of the polyprotein open reading frame must encode glycine, without which infectious virus production cannot proceed. In summary, our data indicate that CVB3 remains viable, even in vivo, in the absence of R-3, and we propose that the most important factor contributing to the high frequency of G+4-not only in CVB but also in other eukaryotic mRNAs, and thus in the consensus motif itself-may be the constraint upon the second amino acid rather than the requirements for translational initiation.

Generation and analysis of an RNA vaccine that protects against coxsackievirus B3 challenge.

Coxsackievirus B3 (CVB3) is an important human pathogen that causes substantial morbidity and mortality but, to date, no vaccine is available. We have generated an RNA-based vaccine against CVB3 and have evaluated it in the murine model of infection. The vaccine was designed to allow production of the viral polyprotein, which should be cleaved to generate most of the viral proteins in their mature form; but infectious virus should not be produced. In vitro translation studies indicated that the mutant polyprotein was efficiently translated and was processed as expected. The mutant RNA was not amplified in transfected cells, and infectious particles were not produced. Furthermore, the candidate RNA vaccine appeared safe in vivo, causing no detectable pathology following injection. Finally, despite failing to induce detectable neutralizing antibodies, the candidate RNA vaccine conferred substantial protection against virus challenge, either with an attenuated recombinant CVB3, or with the highly pathogenic wt virus.

Differential rescue of poliovirus RNA replication functions by genetically modified RNA polymerase precursors.

We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus.

The viral RNA-dependent RNA polymerase (3D(pol)) is highly conserved between the closely related enteroviruses poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3). In this study, we generated PV1/CVB3 chimeric polymerase sequences in the context of full-length poliovirus transcripts to determine the role of different subdomains within the RNA-dependent RNA polymerase of PV1 that are required for functions critical for RNA replication in vitro and in cell culture. The substitution of CVB3 sequences in the carboxy-terminal portion (thumb subdomain) of the polymerase resulted in transcripts incapable of RNA replication. In contrast, three of the seven chimeras were capable of synthesizing RNA, albeit to reduced levels compared to that of wild-type PV1 RNA. Interestingly, one of the replication-competent chimeras (CPP) displayed an inability to generate positive strands, indicating the presence of amino-terminal sequences within the 3D polymerase and/or the 3D domain of the 3CD precursor polypeptide that are necessary for the assembly of strand-specific RNA synthesis complexes. In some constructs, the partial reestablishment of PV1 amino acid sequences in this region was capable of rescuing RNA replication in vitro and in cell culture.

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis.

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.

Subdomain specific functions of the RNA polymerase region of poliovirus 3CD polypeptide.

The 3D polymerase domain of the poliovirus 3CD polypeptide plays a role in modulating its RNA binding and protein processing activities, even though the proteinase catalytic site and RNA binding determinants appear to reside within the 3C(pro) portion of the molecule. In this study, we have generated recombinant 3CD polypeptides that contain chimeric 3D polymerase domains representing suballelic sequence exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) to determine which portions of the 3D domain are responsible for influencing these activities. By utilizing these recombinant protein chimeras in protein processing and RNA binding studies in vitro, we have generated data suggesting the presence of separate subdomains within the polymerase domain of 3CD that may independently modulate its RNA binding and protein processing activities. In predicting where our sequence exchanges map by utilizing the previously published three-dimensional structure of the PV1 3D polymerase, we present evidence that sequences contained within the RNA recognition motif of the polymerase are critical for 3CD function in recognizing the 5' RNA cloverleaf. Furthermore, our protein processing data indicate that at least some of the substrate recognition and processing determinants within the 3D domain of 3CD are separate and distinct from the RNA binding determinants in this domain.