Modification of translational control elements as a new approach to design of attenuated picornavirus strains

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153

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.

Section: Discussionmentioning

confidence: 99%

Evolution of the Sabin Strain of Type 3 Poliovirus in an Immunodeficient Patient during the Entire 637-Day Period of Virus Excretion

Martı́n

Dunn

Hull

et al. 2000

150

153

“…While there is no optimal solution, a number of tissue culture systems have been devised. These include neuroblastoma cell lines that recapitulate neuron-deficient growth of the live attenuated PV vaccines (1,26) and laboratorygenerated attenuated recombinants (2,19,20). However, they are cancerous cells spontaneously interconverting between fibroblast-and neuron-like phenotypes upon prolonged passage (45,47).…”

Section: And 3)mentioning

confidence: 99%

Genetic Adaptation to Untranslated Region-Mediated Enterovirus Growth Deficits by Mutations in the Nonstructural Proteins 3AB and 3CD

Sessions

Dobrikova

Gromeier

2007

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3 terminus is composed of a structured 3UTR and poly ( Enterovirus (EV) plus-strand RNA genome translation and replication are controlled by cis-acting sequence elements mapping to the open reading frame and both 5Ј and 3Ј untranslated regions (UTRs) as well as nonstructural viral gene products. Their ϳ7,400-nucleotide (nt) genomes feature complex structured 5Ј and 3ЈUTRs of ϳ750-and ϳ80-to 110-nt length, respectively, flanking the open reading frame for a single polyprotein (24). The EV 5ЈUTR consists of a ϳ90-nt "cloverleaf" structure serving as a cis-acting replication signal (4) preceding a ϳ450-nt internal ribosomal entry site (IRES) involved in cap-independent translation initiation (23, 39). The 3ЈUTRs of EVs are structurally highly conserved, except coxsackie B viruses (CBVs), whose 3ЈUTRs contain a stem-loop domain (SLD) added to the conventional EV arrangement (42) (see Fig. 1A).The 5Ј cloverleaf and 3ЈUTR are recognized by the same viral proteins with RNA-binding properties and have been implicated in genome replication functions. Viral proteins 3AB (the precursor of 3A, a small membrane-anchoring and RNAbinding protein and 3B, the genome-linked protein VPg) and 3CD (the precursor of the 3C proteinase and the 3D RNAdependent RNA polymerase) interact with these terminal RNA structures (3,15,21,37). Both the cloverleaf and the 3ЈUTR are required for efficient minus-strand synthesis (3,5,27,28,43,50), although viable EVs with 3ЈUTR deletions have been derived (52).In addition to these cis-acting genetic elements and viral RNA-binding proteins, host factors may play a role in the control of EV genome replication and translation. In addition to 3CD and 3AB, the cloverleaf attracts the poly(rC)-binding protein 2 (PCBP2) (15,35). PCBP2 has been proposed to be involved in a switch from translation to replication at plusstrand RNA templates (16). Also, an interaction of PCBP2 with the poly(A)-binding protein has been speculated to mediate template circularization by linking the cloverleaf and poly(A) (22). The complexity of EV translation and RNA synthesis has obstructed unraveling the mechanistic details of their regulation. Several multifunctional viral proteins interact with cisacting signals on both ends on a potentially circular template. Often, the functions of precursor polypeptides are disparate from those of processed viral proteins. For example, although they differ only by the removal of the carboxy-terminal 22 amino acids of 3B, 3AB is believed to costimulate RNA replication by supporting the function of 3CD and 3D, while 3A does not (32,36). cis-acting signals recognized by viral RNAbinding proteins simultaneously contribute to template stability, translation, and/or RNA replication. Moreover, the vir...

“…Apart from host determinants of viral tropism and pathogenicity such as the PV receptor (21), specificity for motor neurons is encrypted within the viral IRES element. This is evident from neuroattenuating mutations mapping to the IRES in the live attenuated (Sabin) vaccine strains of PV (20), as well as from genetically engineered viruses with reduced neurovirulence (2). The most drastic change in neuropathogenicity results from exchange of the entire PV IRES.…”

mentioning

confidence: 99%

Cell-Type-Specific Repression of Internal Ribosome Entry Site Activity by Double-Stranded RNA-Binding Protein 76

Merrill

Dobrikova

Gromeier

2006

Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5 untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-typespecific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells. Posttranscriptional regulation constitutes an important level of control of gene expression. The global rate of protein synthesis is influenced by modification of translation initiation factors. In addition, the translation of many mRNAs is independently regulated by processes targeting their untranslated regions (UTRs). In many instances, specific UTRs bind ribonucleoprotein complexes that alter conditions for translation of individual messages.The plus-strand RNA genomes of Picornaviridae feature complex UTRs involved in the regulation of viral translation and genome replication. Translation of viral RNA occurs in competition with cellular mRNA in infected host cells (3). Accordingly, picornavirus genomes have adopted unconventional features enabling efficient viral translation while limiting host cell protein synthesis. In contrast to cellular mRNAs, picornaviral genomic RNAs are uncapped (34), and their uncommonly large and highly structured 5ЈUTRs contain internal ribosomal entry sites (IRESs) that mediate translation initiation in a 5Ј-end, cap-independent manner (26, 39). IRESmediated translation is unimpeded by virus-induced cleavage of canonical initiation factors eukaryotic initiation factor 4G and poly(A)-binding protein (13,27), indicating that their involvement in initiation at the IRES deviates from capped mRNAs. Moreover, divergent means of translation initiation imply the involvement of noncanonical translation factors or IRES trans-acting factors (ITAFs) (4). Such factors, by virtue of their cell and ...