Abstract
The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)'s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the functional IRES structure.
Highlights
Apart from ribosomal RNAs, internal ribosomal entry sites (IRESs) of many plus strand RNA viruses are the most intriguing RNA structures in biological systems [1], [2], [3], [4], [5], [6], [7]
If the observed tissue and host tropism is related to the quality or quantity of on or more IRES trans-acting factors (ITAFs), over-expression of an ITAF may result in rescue of IRES function of PV1(RIPO) even in neuroblastoma cells or in mouse cells at 37uC
The increase in HRV2 IRESdirected translation in the presence of human PTB 1 (hPTB1) in L20B cells again suggests that the mouse homologue of PTB (mPTB) present in L20B cells is insufficient for optimal function of the HRV2 IRES. These results indicate that the expression of hPTB1 compensates for the intrinsically poor activity of mPTB on HRV2 IRES activity in L20B cells
Summary
Apart from ribosomal RNAs, internal ribosomal entry sites (IRESs) of many plus strand RNA viruses are the most intriguing RNA structures in biological systems [1], [2], [3], [4], [5], [6], [7]. IRESes of plus strand viruses mapping to the 59 non-translated region of the sequence (Fig. 1A) are defined by function, not by structure [8] Their function is to promote initiation of capindependent initiation of translation immediately after the viral genome has entered the cell. ITAFs required for viral IRES function are, surprisingly, of cellular and not of viral origin This makes sense, since an IRES of a plus strand RNA virus must function prior to virus-specific protein synthesis [1], [3]. This notion is supported by the findings from Dobrikova et al [10], where the authors showed that viral IRES function in a properly configured template in uninfected cells IRES efficiency benefit from expression of viral gene products [10]. The most commonly found ITAFs in IRESomes of picornaviruses are polypyrimidine tract-binding protein (PTB) [21], [22], [23], [24], [25], [26], [27], [28]; poly(rC)-binding protein 2 (PCBP2) [29], [30]; the autoantigen La [31], [32], [33], [34], [35]; the upstream of N-ras protein (unr) [24], [25], [36]; and ITAF45 [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
