Biology subjects

Lindenbach, B. D.

Publications and source records attributed to Lindenbach, B. D..

5 recordsLinked to original sources

bioRxiv2023

Astrovirus replication is dependent on induction of double membrane vesicles through a PI3K-dependent, LC3-independent pathway

Human astrovirus is a positive sense, single stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double membrane vesicles (DMVs). Here we show that astrovirus infection leads to an increase in DMV formation, and this process is replication-dependent. Our data suggest that astrovirus infection induces rearrangement of endoplasmic reticulum fragments, which may become the origin for DMV formation. Transcriptional data suggested that formation of DMVs during astrovirus infection requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Inhibition of the PI3K complex leads to significant reduction in viral replication and release from cells. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. ImportanceThese studies provide critical new evidence that astrovirus replication requires formation of double membrane vesicles, which utilize class III PI3K, but not LC3 conjugation autophagy machinery for biogenesis. These results are consistent with replication mechanisms for other positive sense RNA viruses. This suggests that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive sense RNA virus infections.

microbiology↗

bioRxiv2021

Essential Role of Placenta Derived Interferon Stimulated Gene 20 Against ZIKA Virus Infection

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and leads to adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterized the IFN{beta}-mediated antiviral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication; and determined whether we could use the components of the IFN-induced response as a replacement therapy for ZIKA viral infection during pregnancy. We identified and characterized interferon stimulated gene 20 (ISG20), playing a central role in controlling Zika infection in trophoblast cells, and successfully established a recombinant ISG20-Fc protein that effectively decrease viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying immune modulatory functions. Therefore, rISG20-Fc is a promising anti-viral/immune modulatory approach for the control and prevention of RNA viral infections such as ZIKA virus.

immunology↗

bioRxiv2020

Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed -1 ribosomal frameshifting (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting -1 PRF as an effective antiviral strategy for SARS-CoV-2.

molecular biology↗

bioRxiv2019

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the post-fusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFV{Delta}SK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFV{Delta}SK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E expression; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. YFV{Delta}SK/Nluc gene expression was also shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, as well as valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a post-fusion, pre-translation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a post-fusion step in virus entry.\n\nIMPORTANCEFlaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here we show that the yellow fever virus nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

microbiology↗

bioRxiv2018

Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection

The detection of nucleic acids by pattern recognition receptors is an ancient and conserved component of the innate immune system. Notably, RNA virus genomes are sensed by mammalian cytosolic RIG-I-like receptors, thereby activating interferon-stimulated gene (ISG) expression to restrict viral replication. However, recent evidence indicates that the cGAS-STING DNA sensing pathway also protects against RNA viruses. So far, the mechanisms responsible for DNA sensing of RNA viruses, which replicate without known DNA intermediates, remain unclear. By using cGAS gene knockout and reconstitution in human and mouse cell cultures, we discovered that DNA sensing and cGAMP synthase activities are required for cGAS-mediated restriction of vesicular stomatitis virus and Sindbis virus. The level of cGAMP produced in response to RNA virus infection was below the threshold of detection, suggesting that only transient and/or low levels of cGAMP are produced during RNA virus infections. To clarify the DNA ligands that activate cGAS activity, we confirmed that cGAS binds mitochondrial DNA in the cytosol of both uninfected and infected cells; however, the amount of cGAS-associated mitochondrial DNA did not change in response to virus infection. Rather, a variety of pre-existing cytosolic DNAs, including mitochondrial DNA and endogenous cDNAs, may serve as stimuli for basal cGAS activation. Importantly, cGAS knockout and reconstitution experiments demonstrated that cGAS drives low-level ISG expression at steady state. We propose that cGAS-STING restricts RNA viruses by promoting a preparatory immune activation state within cells, likely primed by endogenous cellular DNA ligands.\n\nImportanceMany medically important RNA viruses are restricted by the cGAS-STING DNA-sensing pathway of innate immune activation. Since these viruses replicate without DNA intermediates, it is unclear what DNA ligand(s) are responsible for triggering this pathway. We show here that cGASs DNA binding and signaling activities are required for RNA virus restriction, similar to the mechanisms by which it restricts DNA viruses. Furthermore, we confirmed that cGAS continuously binds host DNA, which was unaffected by RNA virus infection. Finally, cGAS expression correlated with the low-level expression of interferon-stimulated genes in uninfected cells, both in vitro and in vivo. We propose that cGAS-mediated sensing of endogenous DNA ligands contributes to RNA virus restriction by establishing a baseline of innate immune activation.

immunology↗