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This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation …
Biology Articles » Molecular Biology » Protein synthesis in eukaryotes: The growing biological relevance of cap-independent translation initiation » IRESs and viral replication
IRESs and viral replication
- Protein synthesis in eukaryotes: The growing biological relevance of cap-independent translation initiation
Viruses are obligate intracellular parasites and depend on cells for their replication. However, they have evolved mechanisms to ensure that their replication can be achieved in an efficient and, in some instances, a cell-type-specific manner. Yet during the early stages of infection, viral mRNA must compete with their host counterparts for the protein synthesis machinery, not for ribosomes as much as for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs (201, 245). To circumvent this competition, we have described how viruses often modify certain eIFs within infected cells so that ribosomes can be recruited selectively to viral mRNAs. We also have outlined that this strategy implies that such viral mRNAs contain structural features such as the IRES that are distinct from most polymerase II-derived host mRNAs (116, 130, 204, 233). In the following section, we will discuss the relevance of viral IRESs in virus tropism and control of the viral replication cycle.
Factors related to both the host and the infectious agents determine pathogenesis of virus-induced disease. In this sense, there is significant genetic evidence that IRESs contain determinants of cell specificity supporting the notion that viral tropism can be modulated at the level of viral protein
synthesis. In the case of poliovirus (PV), the efficiency of viral mRNA translation is a major determinant of neurovirulence and disease pathogenesis (160-162, 255). In about 1% of humans infected with PV the neurovirulent phenotype is expressed, resulting in paralytic poliomyelitis. Repeated passages of PV strains in animals and cultured cells generated the corresponding attenuated vaccine strains (Sabin types 1-3). Thus, the improved ability of these PV variants to grow in non-nervous tissue compromised their ability to grow in the nervous system, as demonstrated by the decreased neurovirulence in monkeys (162). The live, attenuated Sabin vaccine strains of PV were shown to contain single point mutations within the IRES resulting in compromised translation efficiency specifically in neuronal cells (7, 32, 161, 162, 165, 182, 253, 272). This reduction is mediated by impaired binding of eIF4G, eIF4B and PTB to the IRES leading to an impaired association of ribosomes with the viral RNA (183). In agreement with these findings, the reversion of the Sabin strains towards a pathogenic phenotype, a major cause of vaccine-associated paralytic poliomyelitis, is associated with compensatory mutations in the IRES with a concomitant recovery in secondary structure and translational activity (61, 75, 165).
The ability of PV to adapt to different cell types also correlates with IRES-specific mutations. Most PV strains only infect primates. Since transgenic mice are made PV sensitive by introducing the human PV receptor into their genome, it was assumed that the host range of PV was primarily determined by a cell surface molecule that functions as virus receptor (58, 107). However, the PV-sensitive transgenic (PV-Tg) mouse model led to the characterization of a number of adaptive mutations which allowed PV to replicate in primate cells and the central nervous system (CNS) of monkeys but not in mouse cells or in the CNS of Tg mice (277). The failed capacity to replicate in both PV-Tg mice and in mouse cells was due to an impediment at the level of translation initiation, suggesting that not only the viral receptor but also interactions between the viral IRES and host factors are important determinants of virus host specificity (241). Further genetic evidence correlating IRES activity with virus cell specificity came from the study of a mutant poliovirus in which the IRES had been substituted by the rhinovirus IRES. This chimeric virus replicated as well as wild-type poliovirus in HeLa cells, but replication of the mutant (but not wild-type) viruses was completely restricted in neuronal cells (85).
Another example is the Hepatitis A virus (HAV), sole member of the hepatovirus genus of the picornaviridae. HAV is characterized by its lack of sequence relatedness with other members of the picornavirus family and by several unique biological characteristics, including slow non-cytopathic growth in cell culture and an inability to shut down host-cell protein synthesis (20, 51). HAV possesses an IRES in its 5' UTR, and translation is the rate-limiting step for virus replication (28, 29, 67). However, and in sharp contrast to the major types of picornavirus IRESs, the activity of the HAV IRES requires intact eIF4F (4, 20, 23). In common with PV, highly replicating HAV was recovered following successive passage in cells that normally allowed poor virus replication only. HAV was shown to have acquired mutations in its IRES that enhance replication by facilitating cap-independent translation in a cell-type-specific fashion (33, 65, 66, 234). Interestingly, passage of HAV in different cell types engendered different sets of mutations; however, all adaptive events were clustered within the IRES (33, 65, 66, 234).
The activity of the HCV IRES also varies depending on the cell type (129, 151, 276), and studies comparing the efficiency of the IRES element from different HCV genotypes have established differential translation initiation capabilities (31, 41, 104, 105). Interestingly, the biological differences among HCV genotypes in terms of quantity of virus in serum or sensitivity to antiviral drugs directly correlates with the translation efficiency of the IRES (31). Furthermore, recent studies correlated in vivo tropism of HCV with the ability of the viral IRES to support translation initiation (276). Taken together, these observations support the notion of IRES-dependent virus tropism and stress the role of IRESs in virus pathogenesis.
Upon establishment of a competent infection, IRESs also can play other pivotal roles during viral replication. For example, positive-stranded IRES containing viruses such as PV and HCV utilize the genomic RNA (gRNA) as a common template for translation and RNA replication. Both processes cannot occur simultaneously on a unique RNA template, as they proceed in opposing directions. Consequently, the viral polymerase is unable to use the gRNA as a template for RNA synthesis, while it is being used by translating ribosomes (73, 74, 280). Modulation of IRES activity by viral and cellular factors is required to coordinate these two antagonistic processes. In PV, the binding of the cellular protein PCBP to the IRES enhances viral translation, while the binding of the viral protein 3CD represses translation and facilitates negative-strand synthesis (73, 74). In HCV, the viral core protein down-regulates translation allowing initiation of viral RNA transcription (280). A similar mechanism directed at modulating translation and gRNA encapsidation also has been proposed for retroviruses (26, 48, 184), and a correlation between inhibition of translation and gRNA encapsidation has been reported for the Rous sarcoma virus (RSV) (16, 246). Even though the case for complex retroviruses such as HIV-1 has not been demonstrated experimentally, similar phenomena may be at work, as supported by a number of studies that clearly establish that the full-length HIV-1 5' leader region that contains the IRES element can adopt two mutually exclusive secondary structures (the branched multiple hairpin conformation, BMH, and the long-distance conformation, LDI) that may be functionally different (1). Interestingly, the two conformations differ in their ability to form RNA dimers, structures required for gRNA encapsidation. Moreover, the RNA region, including the first start codon, folds differently in each of these conformations. This region forms an extended hairpin structure in the LDI conformation while creating a long-distance interaction with upstream sequences that occludes the start codon of the viral protein open reading frame in the BMH conformation (1). The conformational switch from LDI to BMH would be favored by the viral protein Gag. This model has been reviewed recently by Darlix et al. (48).
Enormous efforts have been directed at understanding the mechanism underlying viral IRES-mediated translation initiation and the involvement of these elements in virus replication. A better knowledge of the mechanism by which viral-IRES activity is regulated may lead to the design and validation of drug candidates that specifically inhibit virus replication by targeting translation initiation. In the case of HCV, this notion already has received attention (50, 69, 124). Indeed, a number of reports have described specific HCV IRES inhibitors (92, 122, 164, 229). Moreover, at least one phase I dose-escalation clinical study using an HCV IRES inhibitor has been reported (244). Protein synthesis inhibitors are well known in antibacterial therapy, however, to date no antiviral agents have been identified that target viral protein synthesis despite the fact that several viruses of extreme medical significance (e.g. HCV and HIV) possess unique cis-acting RNA elements, such as IRESs. that are essential for mRNA translation. Therefore, understanding the molecular mechanisms underlying viral IRES function will prove instrumental in the development of novel antiviral strategies that specifically target viral protein synthesis.
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