An unusual IRES in the herpes simplex virus thymidine kinase gene

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2005; 102(27):9667-72. DOI: 10.1073/pnas.0504132102
Source: PubMed


We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-eIF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single-base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs.

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    • "et al 2001, Grundhoff and Ganem, 2001, Low et al 2001). Since then few more IRESs have been identified in DNA viral genomes viz., herpes simplex virus (HSV; Griffiths and Coen 2005), simian virus 40 (SV40; Yu and Alwine 2006), and white spot syndrome virus (WSSV, Han and Zhan 2006). "
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    ABSTRACT: Glycoprotein D (gD) and glycoprotein I (gI) genes of bovine herpes virus 1 (BHV1) are contiguous genes with 141 bp region between the two open reading frames (ORFs). Expression of gD and gI from a bicistronic construct containing complete gD and gI gene has been reported either through internal ribosome entry site (IRES)-like element or through the scanning and leakage model (Mukhopadhyay 2000). We here show by computational and experimental means that gD is expressed solely as bicistronic transcript comprising gD and gI coding region in BHV1-infected cells. gI ORF was also shown to express separately. An IRES-like element was also predicted by IRES predicting software in the middle of the gD coding region; within that region a putative promoter was also identified by promoterscan. The intergenic region between the two ORF showed extensive secondary structure which brings the stop codon of gD very close to start codon of gI gene. gD gene transcript in BHV1-infected cells was solely bicistronic. gI transcript was also present in the BHV1-infected cells but in low copy number. The results indicate that gI is probably transcribed from its own transcript in BHV1-infected cells.
    Journal of Biosciences 12/2012; 37(6):971-977. DOI:10.1007/s12038-012-9258-7 · 2.06 Impact Factor
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    • "). The most well documented DNA viral IRES is that of the Kaposi's sarcoma herpes virus (KSHV; Figure 1; Bieleski and Talbot, 2001) while others include Herpes simplex virus (Griffiths and Coen, 2005) and Marek's disease virus (Tahiri- Alaoui et al., 2009) to name a few. The KSHV IRES is representative of most IRESs in the Herpesviridae family in that it is similar in structure to that of HCV, containing two major stem loops (Beales et al., 2003). "
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    ABSTRACT: Many virus infections and stresses can induce endoplasmic reticulum (ER) stress response, a host self-defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to internal ribosome-entry sites (IRES)-dependent. This switching is largely dependent on the mRNA structure of the 5' untranslated region (5' UTR) and on the particular stress stimuli. Picornaviruses and some other viruses contain IRESs within their 5' UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5' UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation, and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation) of host factors for translation initiation, overproduction of homologous proteins of cap-binding protein eukaryotic initiation factors (eIF)4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.
    Frontiers in Microbiology 03/2012; 3:92. DOI:10.3389/fmicb.2012.00092 · 3.99 Impact Factor
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    • "First discovered in poliovirus (Pelletier and Sonenberg, 1988), IRES are typically large complex RNA structures that permit initiation of translation in a cap-independent manner although a few reports have shown IRES activity from short sequences (Chappell et al., 2000; Owens et al., 2001). A 12 nucleotide sequence was shown to be sufficient for tk IRES activity and initiation did not occur on an AUG codon (Griffiths and Coen, 2005). Thus, similarly to the G-string frameshift signal, the C-chord IRES affects translation via an unusually short sequence in a non-canonical manner. "
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    ABSTRACT: Some of the most successful antiviral agents currently available are effective against herpes simplex virus. However, resistance to these drugs is frequently associated with significant morbidity, particularly in immunocompromised patients. In addition to the clinical implications of drug resistance, the range of biological processes exploited by the virus to attain resistance while maintaining pathogenicity is proving to be surprising. These mechanisms, which include ribosomal frameshifting, induced infidelity of the DNA polymerase, and internal ribosome entry, are discussed.
    Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 09/2011; 14(6):251-9. DOI:10.1016/j.drup.2011.08.003 · 9.12 Impact Factor
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