Maria Gonzalez Orozco

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 ^-/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the co-factor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity that correlated with reduced replication of infectious EBOV. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity. Significance Statement: Ebola virus infection can result in high mortality rates with extreme risk of person-to-person transmission. Sporadic outbreaks in Africa have resulted in thousands of fatal cases, highlighting that there is still insufficient knowledge to develop effective antiviral therapies. Like other viruses, Ebola utilizes the host machinery to replicate. Understanding how viral and host proteins interact can help identifying targets for the rational design of antiviral drugs. Here, we identified interactions between the cellular ubiquitin machinery and the Ebola virus polymerase cofactor protein VP35, which are important for efficient virus replication. We developed an approach to identify and block these virus-host interactions using small chemical compounds, which provides a useful tool to study functional molecular mechanisms and at the same time a potential approach to antiviral therapies.

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), the third most common mycobacterial infection. Virulent M. ulcerans secretes mycolactone, a polyketide toxin. Most observations of M. ulcerans infection are described as an extracellular milieu in the form of a necrotic ulcer. While some evidence exists of an intracellular life cycle for M. ulcerans during infection, the exact role that mycolactone plays in this process is poorly understood. Many previous studies have relied upon the addition of purified mycolactone to cell-culture systems to study its role in M. ulcerans pathogenesis and host-response modulation. However, this sterile system drastically simplifies the M. ulcerans infection model and assumes that mycolactone is the only relevant virulence factor expressed by M. ulcerans. Here we show that the addition of purified mycolactone to macrophages during M. ulcerans infection overcomes the bacterial activation of the mechanistic target of rapamycin (mTOR) signaling pathway that plays a substantial role in regulating different cellular processes, including autophagy and apoptosis. To further study the role of mycolactone during M. ulcerans infection, we have developed an inducible mycolactone expression system. Utilizing the mycolactone-deficient Mul::Tn118 strain that contains a transposon insertion in the putative beta-ketoacyl transferase (mup045), we have successfully restored mycolactone production by expressing mup045 in a tetracycline-inducible vector system, which overcomes in-vitro growth defects associated with constitutive complementation. The inducible mycolactone-expressing bacteria resulted in the establishment of infection in a murine footpad model of BU similar to that observed during the infection with wild-type M. ulcerans. This mycolactone inducible system will allow for further analysis of the roles and functions of mycolactone during M. ulcerans infection.