Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, the production of Zika virus has been successful from CMV promoter-driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued viruses maintained the ability to form plaques and replicate efficiently in commonly used cell lines. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

Hexokinase 2 (HK2) catalyzes the first committed step of glucose metabolism—the conversion of glucose to glucose-6-phosphate—directing carbon flux into an array of metabolic pathways such as glycolysis, pentose phosphate pathways, amino acid biosynthesis, and others. Given its prominent role in glucose metabolism, it is critical we understand the regulation of HK2 to appreciate its role in normal physiological function as well as in disease states like cancers. In this study we sought to establish the ability of myeloid cell leukemia 1 (MCL1) to bind and regulate HK2 via its reverse Bcl-2 homology (rBH3) motifs. We employed a combination of biochemical and metabolic analysis in non-small cell lung cancer (NSCLC) cell models (H1299, A549, and NCI-H23) to establish a fundamental link between apoptosis and metabolic regulation. This demonstrates that MCL1 directly binds and enhances HK2 enzymatic activity through interactions with rBH3 on HK2. Consequently, we observe significant reductions in glucose-derived metabolites and impaired cellular metabolic plasticity with the disruption of the HK2-MCL1 interaction. These findings establish a novel mechanism by which anti-apoptotic proteins can directly regulate glucose metabolism.

Hexokinase 2 (HK2) catalyzes the first committed step of glucose metabolism - the conversion of glucose to glucose-6-phosphate – directing carbon flux into an array of metabolic pathways such as glycolysis, pentose phosphate pathway, amino acid biosynthesis and others. Given its prominent role in glucose metabolism, it is critical we understand the regulation of HK2 to appreciate its role in normal physiological function as well as disease states like cancers. Herein we establish a fundamental link between cell survival mechanism and metabolic regulation by demonstrating myeloid cell leukemia 1 (MCL1) directly binds and enhances HK2 enzymatic activity through interactions with reverse Bcl-2 homology motifs (rBH3) on HK2. Consequently, we observe significant reductions in glucose-derived metabolites and impaired cellular metabolic plasticity with disruption of the HK2-MCL1 interaction. These findings establish a novel mechanism by which anti-apoptotic proteins can directly regulate glucose metabolism.