Small Peptide Inhibitors Disrupt a High-Affinity Interaction between Cytoplasmic Dynein and a Viral Cargo Protein

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.To enter the host cell, a virus must cross several barriers to reach the nucleus. Many viruses hijack the microtubular network to be transported along the cytoplasm (7, 18). Dynein is a microtubular motor protein, part of a large macromolecular complex called the microtubular motor complex. Dynein is involved in early stages of the viral life cycle of diverse infections, the first stage being the intracellular transport of the incoming virus along microtubules. Once transported throughout the cytosol, the virus rapidly gains the perinuclear area or the nucleus, where virus replication takes place. The disruption of microtubules or microtubular motor dynein function impairs the transport of a number of viruses; however, the intrinsic mechanism of this transport is unclear. Also, it has not been firmly established whether there is a common mechanism by which these viruses hijack a component of the microtubular motor complex for this purpose (7). A direct interaction between a given viral protein and cytoplasmic dynein for transport has been reported for HIV, herpes simplex virus, African swine fever virus (ASFV), and rabies virus (4, 14, 22, 25). In adenoviruses, a direct interaction of the viral capsid hexon subunit with cytoplasmic dynein has been described recently (5).One of these viruses, ASFV, which is a large DNA virus, enters the cell by dynamin- and clathrin-dependent endocytosis (12), and its infectivity is dependent on the acidification of the endosome. ASFV protein p54, a major protein of virion membranes, interacts with the light-chain dynein of 8 kDa (DLC8), which allows the transport of the virus to the perinuclear area (4), in a region called the microtubular organizing center (MTOC). In this zone, the virus starts replication in the viral factory, a secluded compartment where newly formed virions assemble (11, 13). By binding DLC8, the virus masters intracellular transport to ensure successful infection. However, due to the complexity of the system, the mechanism of this interaction is still elusive.A variety of names have been used for the subunits of the cytoplasmic dynein complex. A new classification for mammalian cytoplasmic dynein subunit genes based on their phylogenetic relationships has been reported in which the DLC8 gene was named DYNLL1 (26).Light dynein chains are responsible for direct cargo binding in the cell, but how do they select so many different cargos? It is not known whether the mode and site of binding is the same for viral proteins and physiological cargos. Within these multimeric complexes, there are a number of molecules that theoretically could interact with a given viral protein. However, to date viral proteins have been described to bind only light or intermediate dynein chains, such as DLC8 and TcTex1 (4, 5, 8). A candidate viral protein would bind one of the DLC binding domains, which in DLC8 are located between the two dimers of the DLC8 molecule (LysXThrThr). Here, we analyzed this interaction between a viral protein and DLC8 in an attempt to elucidate its requirements and relevance for viral infection.To determine whether this interaction is crucial for viral replication or whether it is just one of a number of alternatives for the virus-host interplay, we analyzed the capacity of a set of inhibitor peptides targeting a determined binding domain of the DLC8 molecule to interfere with viral infection by disrupting dynein interaction with viral p54.