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The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus , the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent K d value of 53 nM for binding to the 5′-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development. [ABSTRACT FROM AUTHOR]

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection. [ABSTRACT FROM AUTHOR]

Dengue virus, an ~10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3' positive- stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions. [ABSTRACT FROM AUTHOR]