Date Published: June 29, 2010
Publisher: Hindawi Publishing Corporation
Author(s): Jason Kerkvliet, Ramakrishna Edukulla, Moses Rodriguez.
The RNA-dependent RNA-polymerase, 3Dpol, is an essential component in the picornavirus genome for the replication of single stranded RNA. However, transgenic expression of 3Dpol in mice has antiviral effects. Here, we discuss the structure and function of 3Dpol during picornavirus replication, we review the evidence and consequence of a host immune response to epitopes in 3Dpol after picornavirus infection, highlight data showing the antiviral effects of transgenic 3Dpol from Theiler’s murine encephalomyelitis virus (TMEV), and discuss potential mechanisms by which 3Dpol is causing this antiviral effect in mice.
Picornaviruses are members of a large family of viruses which are classified into twelve different genera. These viruses can cause acute illness as benign as the common cold to chronic illness like poliomyelitis in humans, and foot and mouth disease in split-hoofed animals. Picornavirus was derived from the word “pico” meaning small and “rna” for the single stranded ribonucleic acid it contains. The RNA genomes of picornaviruses are positive sense and can range between 6.7 to 9.5 kilobases. Picornaviral virions are nonenveloped, however, the RNA genome is encapsulated in an icosahedral protein structure made from four capsid proteins encoded by the virus.
Although the overall amino acid sequences of 3D polymerase between different picornaviruses are not very homologous, the basic overall structure and binding motifs between them are very similar (Table 1). Crystal structures from poliovirus, rhinovirus, and foot and mouth disease virus polymerases showed resolution of all amino acids in 3Dpol and showed that the enzyme has the same overall structure as other DNA and RNA polymerases in that it contains finger, palm, and thumb domains [8–11] (Figure 2). Four motifs in the palm are identical to all other RNA polymerases, and the fifth domain is similar to other RNA dependent, but not DNA dependent polymerases [9, 12, 13]. Furthermore, the amino acid sequence residing in these motifs are highly conserved between different picornaviruses . The amino acids at the N-terminus of 3D polymerase were found to be important in the function of this enzyme. The N-termunis encircles the active site in the palm domain and mutations in the N-termunis have been shown to disrupt 3D polymerase activity [8, 11, 15]. More recently, a coxsackievirus 3D polymerase demonstrated the importance of hydrophobicity at residue 5 where the polymerase was more active with amino acids which were more hydrophobic at this residue . This is important for stabilizing the structure of 3D during a conformational change that occurs at the nucleotide repositioning step.
Until recently, T and B cell-specific responses to 3D polymerase in picornaviral infections had not been characterized. Numerous studies have been done to elicit which viral peptides are recognized by the adaptive immune system, however, most of the peptides important in viral clearance were found to be capsid proteins. Although there are many picornaviruses which may elicit a 3D specific immune response, we will focus on the TO subgroup of TMEV because immune responses to this subgroup has been studied in depth as cerebral infection with this subgroup of TMEV is used in mouse models for demyelinating diseases such a multiple sclerosis in mouse strains that are susceptible to a persistent viral infection in the spinal cord. There are also mice resistant to persistent TMEV infection. Resistant mice have similar acute viral encephalitis in the brain after intracerebral infection, however, the virus is then cleared from the CNS. Using susceptible and resistant mouse strains the genetic factors associated with viral susceptibility map to the H-2D locus of the class I major histocompatability complex [36–38]. Mice containing the H-2b,d,k haplotypes are resistant to persistent infection whereas H-2f,p,q,r,s,v haplotypes are susceptible. Clearance of the virus from the CNS in resistant haplotypes is dependent on a class I restricted CD8+ cytotoxic T lymphocyte (CTL) response, because normally resistant mice that are depleted of CD8+ T cells or lack the expression of beta 2 microglobulin (b2M) become susceptible to persistent viral infection [39–41].
Since 3D polymerase plays such a central role in picornavirus replication many strategies have been devised to inhibit the function of 3D and ultimately slow the replication of picornaviruses. One such strategy is the use of nucleoside analogs, such as ribavirin which increases the error rate of the already low fidelity 3D polymerase into a state of lethal mutagenesis. However, there are frequent side effects because most nucleoside analogs also incorporate into host cell RNA. Also, the virus becomes resistant to treatment. The idea that 3D polymerase is a target for antiviral therapy is logical because proper function of 3D is required for picornaviral replication. However, transgenic expression of 3D having antiviral properties is not as easily explained. Our published data is the first using transgenic 3D mice to show antiviral effects. We originally made 3D transgenic mice to be used as a control transgene to study the effects of tolerance to other picornaviral proteins (VP1, VP2, VP3, etc.). At that time, 3D was not thought to be an important antigen for T cells involved in viral clearance. However, after quantifying viral transcripts in 3D transgenic mice we noticed these mice had 100 to 1000 fold reductions in viral RNA after intracranial infection with TMEV compared to nontransgenic mice or transgenic mice expressing other viral capsid proteins. For this reason, the mechanism behind the antiviral effect of transgenic 3D may produce an “outside the box” explanation that will open new avenues for the way scientists think about antiviral research.
The authors have nothing to disclose.