Construction and Rescue of a DNA-Launched DENV2 Infectious Clone

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

Filovirus Entry: A Novelty in the Viral Fusion World

Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of this entry pathway are highlighted here. Virus entry into cells is initiated by the interaction of the viral glycoprotein1 subunit (GP1) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP1 is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP1 are currently incomplete; however, the processed GP1 specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP1 processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP2 subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses

Filovirus Entry: A Novelty in the Viral Fusion World

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Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals

Overview of the autophagic pathway.

<p>Upon infection, viruses trigger the induction of autophagy through a number of mechanisms. Autophagy regulators (i.e., Beclin-1, UVRAG, and ATG14) function in membrane nucleation to form the double-membraned phagophore, which can be blocked via addition of pharmacological inhibitors (3-MA, spautin-1 [SP-1]). Additional autophagy-related proteins (ATG7 and ATG5) mediate the elongation step, in which the phagophore begins to expand until it closes around the material targeted for degradation by sequestration proteins, such as SQSTM/p62. Inhibition of this event is commonly performed through the expression of small interfering RNAs (siRNAs) targeting the autophagic components involved in this process. The completed autophagosome (AP) is then able to fuse with lysosomes (Lyso) via the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of syntaxin 17 (STX17), SNAP29, and VAMP8. The engulfed contents are then degraded, along with the inner membrane in the newly formed autolysosome (AL), in a process termed autophagic flux. Vesicle acidification inhibitors have been used to block degradation in the AL, given that lysosomal proteases are only active at low pH.</p

ADAP2 associates with membrane ruffles and actin.

<p><b>(A),</b> Selected frames (captured every 10min) from time-lapse movies of U2OS cells expressing GFP-ADAP2. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005150#ppat.1005150.s008" target="_blank">S1 Movie</a> for complete movie. At top, differential interference contrast (DIC). <b>(B),</b> Selected frames (captured every 10min) from time-lapse movies of U2OS cells expressing YFP-actin and DsRed-ADAP2. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005150#ppat.1005150.s009" target="_blank">S2 Movie</a> for complete movie. At top, differential interference contrast (DIC). Newly formed actin-enriched membrane ruffles associated with DsRed-ADAP2 are denoted by white arrows. <b>(C)</b>, Immunofluorescence microscopy for actin (in green) in U2OS cells transfected with ADAP2-V5. Cells were fixed and immunostained ~48hrs post-transfection. Areas of colocalization appear as yellow. White box denotes zoomed image at bottom right of merged image. Xy image shown at top and xzy cross-section shown at bottom (cross-section corresponds to the white line shown in the merged panel). <b>(D),</b> Single particle tracking from time-lapse movies of U2OS cells expressing DsRed-ADAP2 over the period of ~3.5hrs. Three independent vesicles were tracked and are shown in light blue, dark blue, or pink. At top, initial image acquired prior to particle tracking (0hr) and at bottom, image acquired at the conclusion of image acquisition. At left, DIC image. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005150#ppat.1005150.s010" target="_blank">S3 Movie</a> for complete movie.</p

ADAP2 associates with LAMP1-positive lysosomes, but not early or late endosomes.

<p><b>(A-C),</b> Confocal micrographs of U2OS cells transfected with mRFP-EEA1 (A), GFP-Rab7 (B), or mRFP-LAMP1 (C) and either GFP-ADAP2 (A, C) or DsRed-ADAP2 (B). Areas of colocalization appear as yellow. DAPI-stained nuclei are shown in blue. White boxes denotes zoomed areas shown in bottom left (A) or right (B, C) corners. <b>(D),</b> Quantification of the extent of colocalization (as assessed by Pearson’s correlation coefficients) in cells transfected with ADAP2 and the indicated endosomal or lysosomal markers. Data are shown as individual Pearson’s correlation coefficients quantified from individual cells. Coefficients >0 indicated positive colocalization. <b>(E-G),</b> Selected frames (captured every 10min) from time-lapse movies of U2OS cells expressing mRFP-EEA1 (E), GFP-Rab7 (F), or mRFP-LAMP1 (G) and either GFP-ADAP2 (E, G) or DsRed-ADAP2 (F). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005150#ppat.1005150.s014" target="_blank">S7</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005150#ppat.1005150.s016" target="_blank">S9 Movies</a> for complete movies. In G, the newly formed ADAP2-positive vesicles are highly associated with LAMP1 and are denoted by white arrows.</p

ADAP2 restricts DENV entry.

<p><b>(A),</b>Titer of NR-DENV (shown as log FFU/mL) under non-illuminated or illuminated (at 0hr) conditions. <b>(B),</b> Schematic of experiments measuring DENV RNA release using NR-DENV. <b>(C),</b> NR-DENV infection as assessed by RT-qPCR in 293T cells transfected with vector control or ADAP2-V5 under non-illuminated (dark grey) or illuminated at 2hr (light grey) or 0hr (white) post-infection. <b>(D),</b> Confocal micrographs from U2OS cells transfected with vector control (for~48hrs) at 30min following the initiation of DENV entry. Cells were fixed and immunostained with antibodies against EEA1 (in red) and DENV (in green). DAPI-stained nuclei are shown in blue. White box denotes zoomed area shown at right. White arrows denote areas of colocalization. <b>(E),</b> Confocal micrographs from U2OS cells transfected with EGFP-ADAP2 (for~48hrs) at 30min following the initiation of DENV entry. Cells were fixed and immunostained with antibodies against DENV (in red). DAPI-stained nuclei are shown in blue. White box denotes zoomed area shown at right. White arrows denote areas of colocalization. <b>(F),</b> Confocal micrographs from U2OS cells transfected with vector control or EGFP-fused ADAP2 (for~48hrs) at 30min following the initiation of VSV entry. Cells were fixed and immunostained with antibodies against VSV-G (in red). DAPI-stained nuclei are shown in blue. White box denotes zoomed area shown at right. White arrows denote areas of colocalization. <b>(G),</b> Pearson correlation coefficient to assess the colocalization between DENV and EEA1 or ADAP2 and VSV and ADAP2, as indicated Each point represents a unique cell (>20 cells from three independent experiments). Line indicates the mean. Data in (A) and (C) are shown as mean ± standard deviation. *p<0.05 and **p<0.01.</p