Zika virus dumbbell-1 structure is critical for sfRNA presence and cytopathic effect during infection.

All flaviviruses contain conserved RNA structures in the 3' untranslated region (3' UTR) that are important for flavivirus RNA replication, translation, and pathogenesis. Flaviviruses like Zika virus (ZIKV) contain multiple conserved RNA structures in the viral 3' UTR, including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus positive-strand genome replication, but the functional role of the flavivirus DB-1 structure and the mechanism by which it contributes to viral pathogenesis are not known. Using the recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones, termed ZIKV-TL.PK and ZIKV-p.2.5', which disrupt DB-1 tertiary folding. We found that viral positive-strand genome replication of both ZIKV DB-1 mutant clones is similar to wild-type (WT) ZIKV, but ZIKV DB-1 mutants exhibit significantly decreased cytopathic effect due to reduced caspase-3 activation. We next show that ZIKV DB-1 mutants exhibit decreased levels of sfRNA species compared to ZIKV-WT during infection. However, ZIKV DB-1 mutant 3' UTRs exhibit unchanged sfRNA biogenesis following XRN1 degradation in vitro. We also found that ZIKV DB-1 mutant virus (ZIKV-p.2.5') exhibited enhanced sensitivity to type I interferon treatment, and both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality due to tissue-specific attenuated viral replication in brain tissue of interferon type I/II receptor knockout mice. We propose that the flavivirus DB-1 RNA structure maintains sfRNA levels during infection despite maintained sfRNA biogenesis, and these results indicate that ZIKV DB-dependent maintenance of sfRNA levels support caspase-3-dependent, cytopathic effect, type I interferon resistance, and viral pathogenesis in mammalian cells and in a ZIKV murine model of disease. IMPORTANCE The group of viruses termed flaviviruses cause important disease throughout the world and include dengue virus, Zika virus, Japanese encephalitis virus, and many more. All of these flaviviruses have highly conserved RNA structures in the untranslated regions of the virus genome. One of the shared RNA structures, termed the dumbbell region, is not well studied, but mutations in this region are important for vaccine development. In this study, we made structure-informed targeted mutations in the Zika virus dumbbell region and studied the effect on the virus. We found that Zika virus dumbbell mutants are significantly weakened or attenuated due to a decreased ability to produce non-coding RNA that is needed to support infection, support virus-induced cell death, and support escape from the host immune system. These data show that targeted mutations in the flavivirus dumbbell RNA structure may be an important approach to develop future vaccine candidates.

The 21st Annual Meeting of the Rocky Mountain Virology Association.

Nestled within the Rocky Mountain National Forest, 114 scientists and students gathered at Colorado State University's Mountain Campus for this year's 21st annual Rocky Mountain National Virology Association meeting. This 3-day retreat consisted of 31 talks and 30 poster presentations discussing advances in research pertaining to viral and prion diseases. The keynote address provided a timely discussion on zoonotic coronaviruses, lessons learned, and the path forward towards predicting, preparing, and preventing future viral disease outbreaks. Other invited speakers discussed advances in SARS-CoV-2 surveillance, molecular interactions involved in flavivirus genome assembly, evaluation of ethnomedicines for their efficacy against infectious diseases, multi-omic analyses to define risk factors associated with long COVID, the role that interferon lambda plays in control of viral pathogenesis, cell-fusion-dependent pathogenesis of varicella zoster virus, and advances in the development of a vaccine platform against prion diseases. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations.

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication.

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

Accurate design of translational output by a neural network model of ribosome distribution.

Synonymous codon choice can have dramatic effects on ribosome speed and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs. Here, we have modeled this variation in translation elongation by using a feed-forward neural network to predict the ribosome density at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and characterized large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of translation speeds predicted with our model. Levels of the fluorescent protein in budding yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo; that this information can be harnessed to design coding sequences; and that control of translation elongation alone is sufficient to produce large quantitative differences in protein output.