Nicolas Albert Gillet

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) evolution has been marked by the rapid accumulation of mutations, among which cytosine-to-uracil (C-to-U) transitions represent a major proportion of observed genomic changes. These mutations have been proposed to result from the activity of host APOBEC3 cytidine deaminases, innate immune enzymes capable of editing viral RNA. However, the molecular mechanisms underlying APOBEC3 involvement in SARS-CoV-2 biology remain poorly understood. Here, we systematically investigated physical interactions between APOBEC family members and the SARS-CoV-2 proteins using a Gaussia princeps protein complementation assay. Screening of APOBEC family proteins against the viral proteome identified specific interactions between APOBEC3G (A3G) and APOBEC3H (A3H) with the viral nucleocapsid (N) protein. These interactions were validated by co-immunoprecipitation and were found to be conserved across nucleocapsid proteins from all seven human coronaviruses, suggesting conserved structural determinants. Mechanistic analyses revealed that the RNA-binding and oligomerization capacities of A3G and A3H are key for their interaction with the SARS-CoV-2 nucleocapsid. Mapping experiments further showed that the C-terminal domain of N constitutes the minimal interacting region, with stronger binding observed in larger constructs encompassing adjacent regions, indicating cooperative stabilization. Further work will be needed to determine whether A3G and/or A3H can restrict viral replication and whether their interaction with the nucleocapsid allows them to access and mutate the viral genome.

Human adenoviruses (HAdVs) are a large family of viruses comprising more than 100 genotypes distributed across seven species (A to G). Depending on the genotype, they cause respiratory, ocular, urinary tract, or gastrointestinal infections. Despite the considerable diversity within the human adenovirus genus, most of our current knowledge comes from studies on HAdV-C5. The availability of experimental tools, including antibodies and reverse genetics systems, for the study of adenoviruses other than the C5 strain remains very limited. Here, we describe a reverse genetics tool to engineer HAdV-A12, named AdamiForge. HAdV-A12 is phylogenetically distant from HAdV-C5 and differs markedly in its clinical manifestations, warranting the development of dedicated experimental tools. In principle, AdamiForge is similar to the widely used reverse genetics tool for HAdV-C5 known as AdenoBuilder. Briefly, the viral genome is divided into several blocks and cloned into high-copy plasmids. These genomic fragments are released using a restriction enzyme and recombined in vitro by Gibson assembly. The reconstituted genome is then delivered into permissive cells to generate new virions. The main advantage of this approach lies in the fragmentation of the viral genome into small plasmids, which can be easily modified. In addition to the reconstitution of the wild-type virus, we engineered two recombinant viruses expressing modified versions of the viral DNA-binding protein fused either to eGFP or to a FLAG-tag. These modifications compensated for the lack of specific antibodies and enabled successful detection of the HAdV-A12 DBP by both Western blotting and immunofluorescence. IMPORTANCE: Adenoviruses belong to a large family of viruses comprising seven species (A–G) and more than 100 genotypes. Beyond their role as human pathogens, adenoviruses have been extensively studied as vectors in gene therapy, vaccine development, and oncolytic virotherapy. To date, research has largely focused on a limited number of strains. However, strain-specific characteristics can substantially influence both pathogenic potential and vector performance. It is therefore essential to expand research to other genotypes and to develop new tools for the study of less-characterized strains such as human adenovirus A12.

Non-coding RNAs play a significant role in viral infection cycles, with recent attention focused on circular RNAs (circRNAs) originating from various viral families. Notably, these circRNAs have been associated with oncogenesis and alterations in viral fitness. However, identifying their expression has proven more challenging than initially anticipated due to unique viral characteristics. This challenge has the potential to impede progress in our understanding of viral circRNAs. Key hurdles in working with viral genomes include: (1) the presence of repetitive regions that can lead to misalignment of sequencing reads, and (2) unconventional splicing mechanisms that deviate from conserved eukaryotic patterns. To address these challenges, we developed vCircTrappist, a bioinformatic pipeline tailored to identify backsplicing events and pinpoint loci expressing circRNAs in RNA sequencing data. Applying this pipeline, we obtained novel insights from both new and existing datasets encompassing a range of animal and human pathogens belonging to Herpesviridae, Retroviridae, Adenoviridae and Orthomyxoviridae families. Subsequent RT-PCR and Sanger sequencings validated the accuracy of the developed bioinformatic tool for a selection of new candidate viral encoded circRNAs. These findings demonstrate that vCircTrappist is an open and unbiased approach for comprehensive identification of virus-derived circRNAs. Significance Statement Circular RNAs (circRNAs) were revealed to have prominent roles in cellular life in the past decade. They were more recently shown to be expressed by viruses, influencing their infectious cycles and host-pathogen relationship. In this context, viruses that were not previously associated with cellular splicing processes are shown to express circRNAs through unknown mechanisms. These non-canonical circRNAs were already shown to be important in the viral cycle and pathogenesis of the viruses they are encoded from. Here, we propose a bioinformatics pipeline that bypasses the limitations of the existing tools in the identification of viral circRNA. Using this pipeline, we discovered numerous candidates and invite the reader to start its own exploration in the realm of viral encoded circRNAs.

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence of SARS-CoV-2 has been demonstrated by our laboratory and others. The aim of this report is to further characterize the associated cellular responses caused by a pre-alpha SARS-CoV-2 strain on differentiated SH-SY5Y and to prevent its cytopathic effect by using a set of entry inhibitors. The susceptibility of SH-SY5Y to SARS-CoV-2 was confirmed at high multiplicity-of-infection, without viral replication or release. Infection caused a reduction in the length of neuritic processes, occurrence of plasma membrane blebs, cell clustering, and changes in lipid droplets electron density. No changes in the expression of cytoskeletal proteins, such as tubulins or tau, could explain neurite shortening. To counteract the toxic effect on neurites, entry inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, and Spike RBD were co-incubated with the viral inoculum. The neurite shortening could be prevented by the highest concentration of camostat mesylate, anti-RBD antibody, and NRP1 inhibitor, but not by soluble ACE2. According to the degree of entry inhibition, the average amount of intracellular viral RNA was negatively correlated to neurite length. This study demonstrated that targeting specific SARS-CoV-2 host receptors could reverse its neurocytopathic effect on SH-SY5Y.

Human adenoviruses (HAdVs) are a large family of DNA viruses counting more than a hundred strains divided into seven species (A to G). HAdVs induce respiratory tract infections, gastroenteritis and conjunctivitis. APOBEC3B is a cytidine deaminase that restricts several DNA viruses. APOBEC3B is also implicated in numerous cancers where it is responsible for the introduction of clustered mutations into the cellular genome. In this study, we demonstrate that APOBEC3B is an adenovirus restriction factor acting through a deaminase-dependent mechanism. APOBEC3B introduces C-to-T clustered mutations into the adenovirus genome. APOBEC3B reduces the propagation of adenoviruses by limiting viral genome replication, progression to late phase, and production of infectious virions. APOBEC3B restriction efficiency varies between adenoviral strains, the A12 strain being more sensitive to APOBEC3B than the B3 or C2 strains. In A12-infected cells, APOBEC3B clusters in the viral replication centers. Importantly, we show that adenovirus infection leads to a reduction of the quantity and/or enzymatic activity of the APOBEC3B protein depending on the strains. The A12 strain seems less able to resist APOBEC3B than the B3 or C2 strains, a characteristic which could explain the strong depletion of the APOBEC3-targeted motifs in the A12 genome. These findings suggest that adenoviruses evolved different mechanisms to antagonize APOBEC3B. Elucidating these mechanisms could benefit the design of cancer treatments. This study also identifies adenoviruses as triggers of the APOBEC3B-mediated innate response. The involvement of certain adenoviral strains in the genesis of the APOBEC3 mutational signature observed in tumors deserves further study.

The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.