Biology subjects

Pietzsch, C.

Publications and source records attributed to Pietzsch, C..

4 recordsLinked to original sources

bioRxiv2021

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leveraged proximity proteomics and conducted an siRNA screen to define the functional interactome of EBOV polymerase. As proof-of-principle, we validated two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose both GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduced viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

microbiology↗

bioRxiv2021

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed an siRNA screen to identify those interactors that have functional roles in authentic LASV infection. As proof-of-principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses. Significance StatementLassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no FDA-approved therapeutic intervention to treat LF. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome, and thus is an attractive target for antiviral intervention. Here we present the first host interactome of the LASV polymerase, which can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF.

microbiology↗

bioRxiv2021

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

immunology↗

bioRxiv2020

Topoisomerase III-beta is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-{beta} (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

microbiology↗