Kleiner, V. A. | Search bioRxiv

bioRxiv2026

eIF4E3 drives translation of viral mRNAs with short 5' UTRs

Eukaryotic cells express three paralogs of the cap-binding protein eIF4E, yet the functions of the less-studied family members remain poorly understood. Here we demonstrate that eIF4E3, a paralog whose expression is tissue-restricted and whose activity is insensitive to inhibition by eIF4E-binding proteins (4EBPs), drives efficient translation of viral mRNAs with short 5 untranslated regions (UTRs). Many negative-strand RNA viruses (NSVs), including vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), and influenza A virus (IAV), produce mRNAs with extremely short 5UTRs that are incompatible with canonical cap-dependent scanning translation initiation. Using an auxin-inducible degron (AID) system to acutely deplete endogenous eukaryotic initiation factors during active viral infection, we demonstrate that translation of these short-UTR viral mRNAs occurs independently of eIF4E1, the canonical cap-binding protein, while remaining dependent on eIF4E3. In contrast, Ebola virus (EBOV), whose mRNAs bear long, structured 5UTRs, remains eIF4E1-dependent, implicating 5UTR length and structural complexity as cis determinants of eIF4E paralog selectivity. During VSV infection, 4EBP dephosphorylation sequesters eIF4E1 and broadly suppresses host cap-dependent translation. Because eIF4E3 escapes 4EBP-mediated regulation and preferentially engages short, unstructured 5UTRs, it is uniquely positioned to sustain viral protein synthesis under these conditions. These findings reveal that VSV exploits a cellular paralog-switching mechanism by co-opting eIF4E3 to maintain viral translation when canonical eIF4F activity is suppressed and establish eIF4E3 as a proviral factor whose tissue-restricted expression in the lung may influence susceptibility to clinically important respiratory pathogens.

molecular biology↗

bioRxiv2022

DISTINCTIVE FEATURES OF THE RESPIRATORY SYNCYTIAL VIRUS PRIMING LOOP COMPARED TO OTHER NON-SEGMENTED NEGATIVE STRAND RNA VIRUSES

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Author SummaryRSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.

microbiology↗