Initiation Of Negative-strand Synthesis Research Articles

Structural Dynamics of the Dengue Virus Non-structural 5 (NS5) Interactions with Promoter Stem Loop A (SLA).

The dengue virus (DENV) NS5 protein plays a central role in dengue viral RNA synthesis which makes it an attractive target for antiviral drug development. DENV NS5 is known to interact with the stem-loop A (SLA) promoter at the 5'-untranslated region (5'-UTR) of the viral genome as a molecular recognition signature for the initiation of negative strand synthesis at the 3' end of the viral genome. However, the conformational dynamics involved in these interactions are yet to be fully elucidated. Our study explores the structural dynamics of NS5 from DENV serotype 2 (DENV2 NS5) in complex with SLA, employing surface plasmon resonance (SPR), hydrogen - deuterium exchange coupled to mass spectrometry (HDX-MS), computational modeling, and cryoEM single particle analysis to delineate the molecular details of their interaction. Our findings indicate that DENV2 NS5 binds SLA in a closed conformation with significant interdomain cooperation between the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, a feature integral to the interaction. Our HDX-MS studies reveal SLA-induced conformational changes in both domains of DENV2 NS5, reflecting a potential mechanism for dengue NS5's multifunctional role in viral replication. Lastly, our cryoEM structure provides the first visualization of the DENV2 NS5-SLA complex, confirming a conserved SLA binding mode across DENV serotypes. These insights obtained from our study enhance our understanding of dengue NS5's complex conformational landscape, supporting the potential development of antiviral strategies targeting dengue NS5's conformational states.

5′- and 3′-noncoding regions in flavivirus RNA

Publisher SummaryThe flavivirus genome is a capped, positive-sense RNA approximately 10.5 kb in length. It contains a single long open reading frame (ORF), flanked by a 5´ noncoding regions (NCR), which is about 100 nucleotides in length, and a 3´ NCR ranging in size from about 400 to 800 nucleotides in length. The conserved structural and nucleotide sequence elements of these NCRs and their function in RNA replication and translation are the subjects of this chapter. The 5´ and 3´ NCRs play a role in the initiation of negative-strand synthesis on virus RNA released from entering virions, switching from negative-strand synthesis to synthesis of progeny plus strand RNA at late times after infection, and possibly in the initiation of translation and in the packaging of virus plus strand RNA into particles. The presence of conserved and nonconserved complementary nucleotide sequences near the 5´ and 3´ termini of flavivirus genomes suggests that ‘‘panhandle’’ or circular RNA structures are formed transiently by hydrogen bonding at some stage during RNA replication.

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis.

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

Structure-infectivity analysis of the human rhinovirus genomic RNA 3' non-coding region.

The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary structure probing techniques, we have demonstrated the existence of a single stem-loop structure contained entirely within the 3' NCR, which appears to be phylogenetically conserved within the rhinovirus genus. We also report the in vivo analysis of a number of 3' NCR deletion mutations engineered into infectious cDNA clones which were designed to disrupt the stem-loop secondary structure to varying degrees. Large deletions (up to 37 nt) resulted in defective growth phenotypes, although they were not lethal. We propose that the absolute requirements for initiation of negative strand synthesis are less stringent than previously postulated, even though defined RNA secondary structure determinants may have evolved to facilitate and/or regulate the process of viral RNA replication.