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Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state--dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV- induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcy receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections. [ABSTRACT FROM AUTHOR]

The emergence of Zika virus (ZIKV) as a major public health threat has focused research on understanding virus biology and developing a suite of strategies for disease intervention. Recent advances in cryoelectron microscopy have accelerated structure-function studies of flaviviruses and of ZIKV in particular. Structures of the mature and immature ZIKV have demonstrated its similarity with other known flaviviruses such as dengue and West Nile viruses. However, ZIKV's unique pathobiology demands an explanation of how its structure, although similar to its flavivirus relatives, is sufficiently unique to address questions of receptor specificity, transmission, and antigenicity. Progress in defining the immunodominant epitopes and how neutralizing antibodies bind to them will provide great insight as vaccines progress through clinical trials. Identification of host receptors will substantially illuminate the interesting ZIKV tropism and provide insights into pathogenesis. Although the answers to all of these questions are not yet available, rapid progress in combining structural biology with other techniques is revealing the similarities and the differences in virion structure and function between ZIKV and related flaviviruses. [ABSTRACT FROM AUTHOR]

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies. [ABSTRACT FROM AUTHOR]

The recent rapid spread of Zika virus and its unexpected linkage to birth defects and an autoimmune neurological syndrome have generated worldwide concern. Zika virus is a flavivirus like the dengue, yellow fever, and West Nile viruses.We present the 3.8 angstrom resolution structure of mature Zika virus, determined by cryo-electron microscopy (cryo-EM).The structure of Zika virus is similar to other known flavivirus structures, except for the ~10 amino acids that surround the Asn154 glycosylation site in each of the 180 envelope glycoproteins that make up the icosahedral shell. The carbohydrate moiety associated with this residue, which is recognizable in the cryo-EM electron density, may function as an attachment site of the virus to host cells. This region varies not only among Zika virus strains but also in other flaviviruses, which suggests that differences in this region may influence virus transmission and disease. [ABSTRACT FROM AUTHOR]

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. [ABSTRACT FROM AUTHOR]

• Sanofi Pasteur's tetravalent dengue vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1–4). • Potential link between CYD vaccine virus maturity and clinical trial efficacy. • Maturity assessment of four CYD vaccine viruses by mass spectrometry. • Maturity assessment of four CYD vaccine viruses by cryo-electron microscopy. • CYD dengue vaccine viruses lots used in recent phase III efficacy trials, primarily display a mature phenotype. Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1–4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype. [ABSTRACT FROM AUTHOR]

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1H-NMR, 13C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC50 value of 160 nM. Compound 13b exhibited an enzyme IC50 value of 4.9 µM. However, it exhibited a potent antiviral EC50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site. [ABSTRACT FROM AUTHOR]