Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes cellular trafficking pathways to process its structural proteins and move them to the site of assembly. Nevertheless, the exact process of assembly and subcellular trafficking of SARS-CoV-2 proteins remains largely unknown. Here, we have identified and characterized Rab1B as an important host factor for the trafficking and maturation of the spike protein (S) after synthesis at the endoplasmic reticulum (ER). Using confocal microscopy, we showed that S and Rab1B substantially colocalized in compartments of the early secretory pathway. Co-expression of dominant-negative (DN) Rab1B N121I leads to an aberrant distribution of S into perinuclear spots after ectopic expression and in SARS-CoV-2-infected cells caused by either structural rearrangement of the ERGIC or Golgi or missing interaction between Rab1B and S. Western blot analyses revealed a complete loss of the mature, cleaved S2 subunit in cell lysates and culture supernatants upon co-expression of DN Rab1B N121I. In sum, our studies indicate that Rab1B is an important regulator of trafficking and maturation of SARS-CoV-2 S, which not only improves our understanding of the coronavirus replication cycle but also may have implications for the development of antiviral strategies.

Targeted protein degradation represents a new paradigm in drug development. By hijacking the cellular ubiquitin-proteasome-system pathogenic proteins are degraded via heterobifunctional molecules, referred to as proteolysis targeting chimeras (PROTACs). However, to date, only few PROTACs targeting viral or proviral proteins have been developed. To explore the possibilities and advantages of antiviral PROTACs, we have developed an antiviral PROTAC against the SARS-CoV-2 main protease (M ^Pro ) using an allosteric warhead. Here, we present the design of ten M ^Pro degraders that were developed using pelitinib as a warhead, an allosteric binder of M ^Pro . Among several candidates, LLP019 emerged as the most potent molecule, capable of degrading up to 90% of M ^Pro after ectopic expression in HEK293F cells. LLP019 displayed significant antiviral activity against several variants of concern of SARS-CoV-2 in infected Calu3 cells. In conclusion, we show that the development of antiviral PROTACs using an allosteric warhead represents a promising antiviral strategy, expanding the range of possible target proteins and ligands. SYNPOSIS: We present a proof-of-concept study to construct a PROTAC targeting the SARS-CoV-2 main protease (M ^Pro ) using a warhead that binds outside the catalytic pocket.

Viruses employ cellular host factors not only for replication and transcription but also for transport and assembly. Rab-GTPases, as master regulators of intracellular trafficking pathways, are commonly involved host factors and useful tools for identification of transport pathways used but certain viruses. Here, we present Rab1B as an essential host factor for trafficking and maturation of the SARS-CoV-2 spike protein.

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40?s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention.