Building Blocks of Understanding: Constructing a Reverse Genetics Platform for studying determinants of SARS-CoV-2 replication. (preprint)

To better understand viral pathogenesis, host-virus interactions, and potential therapeutic interventions, the development of robust reverse genetics systems for SARS-CoV-2 is crucial. Here, we present a reverse genetics platform that enables the efficient manipulation, assembly, and rescue of recombinant SARS-CoV-2. The versatility of our reverse genetics system was demonstrated by generating recombinant SARS-CoV-2 viruses. We used this system to generate N501Y and Y453F spike protein mutants. Characterization studies revealed distinct phenotypic effects, impact on viral fitness, cell binding, and replication kinetics. We also investigated a recently discovered priming site for NSP9, which is postulated to produce a short RNA antisense leader sequence. By introducing the U76G mutation into the 5-UTR, we show that this priming site is necessary for the correct production of genomic and subgenomic RNAs, and also for efficient viral replication. In conclusion, our developed reverse genetics system provides a robust and adaptable platform for the efficient generation of recombinant SARS-CoV-2 viruses for their comprehensive characterization.

COMPASS – rapid and highly sensitive medical point-of-care diagnostic (preprint)

In the last decade Magnetic nanoparticles (MNPs) have gained an enormous interest in specialized areas such as medicine, cancer theranostics, biosensing, catalysis, agriculture, and the environmental protection. By controlled engineering of specific surface properties, named functionalization, MNPs are gaining special features for desired applications, e.g., bioassays for the detection of biomolecules or biomarkers such as antibodies. The characterization as well as a highly specific measurement of such binding states is of high interest and limited to highly sensitive techniques such as ELISA (Enzyme-linked Immunosorbent Assay) or flow cytometry, which are relatively inflexible, difficult to handle, expensive and time-consuming. Novel upcoming methods, such as ACS (AC susceptometry) or MPS (Magnetic Particle Spectroscopy), exploit the magnetization response of functionalized MNP ensembles to assess specific information about the MNP mobility within their environment as well as the conjugations of chemical or biological compounds on their surface. Both methods have shown promising results reaching similar sensitivities within short measurement times but showing difficulties in data interpretation. Here, we report a novel method, COMPASS (Critical Offset Magnetic PArticle SpectroScopy), which is based on a critical offset magnetic field of MNPs, which enables sensitive detection to minimal changes in mobility of MNP ensembles, e.g., resulting from SARS-CoV-2 antibodies binding to the S antigen on the surface of functionalized MNPs. With a validated sensitivity of 0.85 fmole/50 µl sample volume ( ≙ 33 pM) SARS-CoV-2-S1 antibodies, measured with a low-cost portable COMPASS device, the proposed technique is not only competitive with the sensitivity of commonly used ELISA or flow cytometry methods but provides more flexibility, robustness and rapid measurement withinwell below a minute per sample, including sample conjugation, mixing and incubation times. The underlying physical effect is based on an offset magnetic field induced suppression of a higher harmonic in the nonlinear magnetization response of the MNP to a time varying magnetic field resulting in a highly sensitive response of the signal phase to minimal changes in particle mobility. Since this effect is independent of MNP concentration, the sample handling is much simpler and robust. Our method thus may pave the way for deeper insights into complex and rapid binding dynamics of functionalization chemistry and can lead to a huge step forwards in point-of-care diagnostics as well as impacts other fields in research and industries.

Selective inhibition of microRNA processing by a herpesvirus-encoded microRNA triggers virus reactivation from latency (preprint)

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the latent-lytic switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture, which impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 was sufficient to trigger virus reactivation from latency thereby identifying it as a readily drugable master regulator of the herpesvirus latent-lytic switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders like myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and Long-COVID.