The Internal Ribosome Entry Site of Dengue Virus mRNA Is Active When Cap-Dependent Translation Initiation Is Inhibited

Abstract

Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Translation initiation of the DENV mRNA can occur following a cap-dependent or a cap-independent mechanism. Two non-mutually exclusive cap-independent mechanisms of translation initiation have been described for the DENV mRNA. The first corresponds to a 5′end-dependent internal ribosome entry site (IRES)-independent mechanism, while the second relies on IRES-dependent initiation. In this report, we study the recently discovered DENV IRES. Results show that the DENV IRES is functional in the rabbit reticulocyte (RRL) in vitro translation system. In accordance, the activity of DENV IRES was resistant to the cleavage of eIF4G by the Foot-and-mouth disease virus leader protease in RRL. In cells, the DENV IRES exhibited only a marginal activity under standard culture conditions. The DENV IRES showed weak activity in HEK 293T cells; however, the DENV IRES activity was significantly enhanced in HEK 293T cells expressing the Human rhinovirus 2A protease. These findings suggest that the DENV IRES enables viral protein synthesis under conditions that suppress canonical translation initiation. IMPORTANCE Dengue virus (DENV), the etiological agent of Dengue, a febrile and hemorrhagic disease, infects millions of people per year in tropical and subtropical countries. When infecting cells, DENV induces stress conditions known to inhibit canonical protein synthesis. Under these conditions, DENV mRNA thrives using non-canonical modes of translation initiation. In this study, we characterize the mechanism dependent upon an internal ribosome entry site (IRES). Herein, we describe the activity of the DENV IRES in vitro and cells. We show that in cells, DENV IRES enables the viral mRNA to translate under conditions that suppress canonical translation initiation.

PKR-mediated Stress Response Enhances Dengue and Zika Virus Replication

“…Using DENV and ZIKV, this mechanism was later shown to be mediated by an internal ribosomal entry site (IRES localized in viral -UTR [4]. A recent report has confirmed the existence of the IRES element [5], indicating, along with previous suggestions [3,6,7] that a cap-dependent translation would be used for viral polyprotein synthesis until the suppression of canonical translation initiation when a switch to cap-independent translation would take place. This alternative cap-independent mechanism, including cell conditions to promote the switch in translation mode, remains to be fully characterized.…”

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs

“…The non-canonical mechanism of translation initiation is undoubtedly characteristic of mRNAs of the Flaviviridae family members: Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), and the aforementioned DENV. Although these mRNAs are capped, their efficient translation continues after inactivation of eIF4E and eIF4G, and the presence of a functional cap is not necessary [ 25 , 174 , 175 ]. According to some data, this property is completely determined by the 5′ UTR of viral mRNA [ 174 , 175 ], while according to others, it also requires interaction of 5′ and 3′ UTR [ 25 ], yet the above-described cHP hairpin in the coding part is not required for this.…”

“…Although these mRNAs are capped, their efficient translation continues after inactivation of eIF4E and eIF4G, and the presence of a functional cap is not necessary [ 25 , 174 , 175 ]. According to some data, this property is completely determined by the 5′ UTR of viral mRNA [ 174 , 175 ], while according to others, it also requires interaction of 5′ and 3′ UTR [ 25 ], yet the above-described cHP hairpin in the coding part is not required for this. Although in 2006 it was demonstrated [ 25 ] that the 5′ UTR of the DENV mRNA had no IRES activity, two recent studies [ 174 , 175 ] dispute this statement.…”

“…According to some data, this property is completely determined by the 5′ UTR of viral mRNA [ 174 , 175 ], while according to others, it also requires interaction of 5′ and 3′ UTR [ 25 ], yet the above-described cHP hairpin in the coding part is not required for this. Although in 2006 it was demonstrated [ 25 ] that the 5′ UTR of the DENV mRNA had no IRES activity, two recent studies [ 174 , 175 ] dispute this statement. Taking into account the difficulties described above in the interpretation of the results obtained using bicistronic reporters, additional studies would help resolve this issue.…”

RNA-Binding Proteins at the Host-Pathogen Interface Targeting Viral Regulatory Elements

“…In contrast, it has been recently found that cap-dependent translation initiation and cap-independent translation initiation mechanisms alternate in DENV infected cells. Interestingly, the weak activity of DENV IRES-like was enhanced in cells expressing the rhinovirus 2A protease, suggesting that the DENV IRES activity enables viral protein synthesis under conditions that suppress cap-dependent translation initiation [ 38 ].…”