An RNA Thermometer Activity of the West Nile Virus Genomic 3′-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching

Abstract

The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was proposed to act as a replication silencer. The lower part of the 3′SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3′SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3′SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3′SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3′SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3′SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-Coding RNAs

“…Without detailed information on flaviviral nucleic acid tertiary structures, it will be challenging to obtain a complete picture of how flaviviral TRs play a part in viral replication. Recently, it was shown for the West Nile virus that the 3' stem-loop region undergoes a temperature-dependent genome cyclization, meaning that the higher temperatures in humans facilitate genome cyclization and viral replication, which does not occur in mosquitos due to their decreased body temperature [72]. Potential temperature-based change in viral RNA tertiary structure is an important aspect to investigate further.…”

Information Encoded by the Flavivirus Genomes beyond the Nucleotide Sequence

“…Two conserved base pairs can be also found at the base of the 3′SL element in the JEV and DENV clades, which are flanked by a metastable structure identified as two symmetrical bulges ( Figure 2 ) [ 58 , 59 ]. This region stabilizes the whole 3′SL structure, and it has been proposed to be involved in the switch from the linear to the cyclized genome [ 59 , 60 ]. Indeed, the 5′ basal segment of the 3′SL contains the 3′UAR motif, which is essential for genome cyclization ( Figure 2 ) ([ 44 , 60 ]; see below).…”

“…Indeed, the 5′ basal segment of the 3′SL contains the 3′UAR motif, which is essential for genome cyclization ( Figure 2 ) ([ 44 , 60 ]; see below). The unwinding of this segment is required for the efficient replication of MBFV in mammalian cells, while in mosquito cells, such structural destabilization occurs only marginally (see below; [ 59 ]).…”

“…The formation of the so-called UAR pair requires the unwinding of the UFS at the 5′UTR, which is likely mediated by cellular factors such as AUF-1 [ 58 ]. Destabilization at the base of 3′SL within the 3′UTR is also needed ( Figure 1 and Figure 2 ) [ 12 , 13 , 59 , 80 ].…”

Siblings or doppelgängers? Deciphering the evolution of structured cis-regulatory RNAs beyond homology

“…RNA thermometers (RNATs) involved in host-switching are well known in bacteria [48], but an RNAT with a similar host-switching function was recently identified in a flavivirus. According to researchers, West Nile virus has a 3 0 RNAT to aid in the switching between cold-blooded and warm-blooded hosts [49]. This viral RNAT was found to alter the circularization rate, and thus the replication rate of the virus, allowing for persistent, low-level infection in insects and acute, high-level infection in warm-blooded hosts.…”