A noncanonical mechanism of DENV translation initiation

While DENV and other flaviviruses containing capped RNA genomes undergo cap-dependent translation, DENV has been shown to translate efficiently under circumstances in which cap-dependent cellular translation is suppressed through the depletion of the cap-binding protein, eIF4E (Edgil et al 2006). To investigate this phenomenon, we used several approaches to inhibit cap-dependent translation. Initially, the compounds rapamycin, wortmannin and LY294002 were used to suppress cellular translation initiation through the sequestration of eIF4E. The downstream effect of the interaction of rapamycin with the mammalian Target of Rapamycin (mTOR) is the hypo-phosphorylation of the eIF4E-binding protein 1 (4E-BP) (Vanhaesebroeck et al 2001). Similarly, wortmannin and LY294002 inhibit the phosphoinositol-3-kinase (PI3-kinase) pathway, which also results in the hypo-phosphorylation of 4E-BP (Vanhaesebroeck et al 2001). In its hypo-phosphorylated form, 4E-BP binds to and sequesters eIF4E from the eIF4F translation initiation complex, thus inhibiting cap-dependent translation. The eIF4F complex consists of eIF4E, the scaffold protein eIF4G and the helicase eIF4A, and is necessary for recruitment of the 43S ribosomal complex to the 5'-terminus of the mRNA (Gale et al 2000). The impact of treatment with these drugs on translation of cellular cap-dependent mRNAs was compared to their effect on DENV reporter constructs and on viral replication. When cells were incubated with DENV in the presence of rapamycin, LY294002 or wortmannin, viral titres

1,000,000

100,000

10,000

1,000

100,000

10,000

Untreated Wortmannin LY294002 1uM 40uM

40uM LY294

% protein expression vs. untreated control:

40uM LY294

% protein expression vs. untreated control:

FIG. 5. DENV replication and translation are resistant to inhibitors of cap-dependent translation. (A) DENV replicates in cells exposed to inhibitors for 24 h. BHK cells (2 X 105) were exposed to DENV-2 strain 16681 and simultaneously treated with 40 nM LY294002 or 1 nM wortmannin per well. Cells were incubated for 24 h at 37°C. Cell supernatants were collected and infectious virus titered using BHK21 cells (PFU/mL). The data are expressed as an average of three experiments. Error bars indicate standard deviation. (B) DENV RNA is translated in inhibitor-treated cells. Cells were treated with LY294002 or wortmannin as described above. One hour prior to metabolic labelling (12 or 24 h post-infection), cells were starved in cysteine-methionine-free medium in the presence of inhibitors. Cells were then pulsed with 150 nCi of [35S] cysteine-methionine for 30 min, harvested and analysed by SDS-PAGE. DENV NS5 (arrow) and representative cellular proteins were quantitated as percent protein relative to the untreated control. Data are representative of three experiments.

remained unchanged at 24 h post-infection (Fig. 5A). Furthermore, whereas metabolic labelling of total cellular protein in inhibitor-treated cells revealed an inhibition of cellular protein synthesis 12 h post-infection, translation of DENV proteins, represented by the DENV RNA-dependent RNA polymerase (NS5), was relatively unaffected (Fig. 5B).

Other conditions that inhibit cellular cap-dependent translation, such as siRNAmediated depletion of eIF4E, also did not affect the yield of DENV progeny from infectious virus, the translation of viral proteins, or the translation of reporter genes flanked by the DENV 5' and 3' UTRs (Edgil et al 2006). Additionally, translation of a reporter construct containing both the DENV 5' and 3' UTRs required significantly higher concentrations of cap analogue to competitively reduce translation than did constructs containing cellular or viral 5' UTRs and a poly(A) tail. Importantly, cap-independent translation from both non-functionally capped DENV reporter constructs and infectious DENV RNA was triggered when eIF4E was depleted or sequestered (Edgil et al 2006). Under conditions of reduced eIF4E, this non-canonical DENV translation required both the DENV 5' and 3' UTRs for activity and did not proceed via internal ribosome entry but rather by an end-dependent mechanism, as the introduction of stable hairpins at the 5' end inhibited translation initiation (data not shown). We propose a model

FIG. 6. Model of S'-3' interactions in canonical cap-dependent and non-canonical DENV translation initiation. When eIF4E is abundant, a canonical cap-dependent scanning mechanism of translation initiation occurs (left). When eIF4E is limiting, the DENV 3' UTR interacts with host proteins to deliver and/or stabilize key translation initiation factors at the 5' UTR in the absence of eIF4E using a non-canonical mechanism of translation initiation (right).

Canonical translation Non-canonical translation

FIG. 6. Model of S'-3' interactions in canonical cap-dependent and non-canonical DENV translation initiation. When eIF4E is abundant, a canonical cap-dependent scanning mechanism of translation initiation occurs (left). When eIF4E is limiting, the DENV 3' UTR interacts with host proteins to deliver and/or stabilize key translation initiation factors at the 5' UTR in the absence of eIF4E using a non-canonical mechanism of translation initiation (right).

wherein DENV is translated via a canonical cap-dependent mechanism when eIF4E is abundant. However, under cellular conditions where eIF4E is limiting, DENV RNA may undergo a reorganization of viral ribonucleoprotein (RNP) complexes such that RNA structures or sequences in the 3' UTR interact with higher affinity with protein complexes containing eIF4G and eIF4A to deliver key translation initiation factors to the DENV 5' UTR (Fig. 6), similar to the mechanism proposed for cap-independent translation of Luteovirus RNAs, such as the barley yellow dwarf virus genome (Guo et al 2001). This may be critical for DENV survival in differentiated cell types with low levels of eIF4E (Grolleau et al 1999, Krichevsky et al 1999), such as the dendritic cells and monocyte/macrophages that are the targets of DENV infection in humans (Ho et al 2001, Jessie et al 2004, Wu et al 2000).

Conclusions

In summary, flavivirus replication is regulated by sequences and structures in both the coding and non-coding regions of the viral RNA. Cyclization of the genomic RNA is necessary for RNA synthesis and viral replication and is mediated by the CS and UAR sequences at the 5' and 3' ends of the flavivirus RNA. Conserved structures in the 3' UTR, such as the 3'SL, play a role in both viral translation and RNA synthesis. Additionally, a hairpin structure in the capsid coding region regulates translation start site selection as well as another step in viral replication. As more RNA elements and UTR-binding proteins are identified that modulate viral translation and RNA synthesis, a more detailed understanding of the molecular events in flavivirus replication is being obtained.

A ck nowledgments

Funding was provided by the Pew Charitable Trusts (#2617SC) and NIH (AI052324). References

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