Virus and host interactions critical for filoviral RNA synthesis as therapeutic targets

Abstract

Filoviruses, which include Ebola virus and Marburg virus, are negative-sense RNA viruses associated with sporadic outbreaks of severe viral hemorrhagic fever characterized by uncontrolled virus replication. The extreme virulence and emerging nature of these zoonotic pathogens make them a significant threat to human health. Replication of the filovirus genome and production of viral RNAs requires the function of a complex of four viral proteins, the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30) and large protein (L). The latter performs the enzymatic activities required for production of viral RNAs and capping of viral mRNAs. Although it has been recognized that interactions between the virus-encoded components of the EBOV RNA polymerase complex are required for viral RNA synthesis reactions, specific molecular details have, until recently, been lacking. New efforts have combined structural biology and molecular virology to reveal in great detail the molecular basis for critical protein-protein interactions (PPIs) necessary for viral RNA synthesis. These efforts include recent studies that have identified a range of host factors and in some instances demonstrated unique mechanisms by which they act. For a select number of these interactions, combined use of mutagenesis, over-expressing of peptides corresponding to PPI interfaces and identification of small molecules that disrupt PPIs have demonstrated the functional significance of virus-virus and virus-host PPIs and suggest several as potential targets for therapeutic intervention.

Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

“…These data clearly suggest that NP-Ct has an important role in genomic RNA incorporation or stability within the VLP nucleocapsids, and as such defines a novel function for this domain. Our finding that NP(1-641) has wild-type transcription/replication activity in p0 cells is consistent with it containing previously identified binding sites for VP35 and VP30 that support transcription/replication, as well as the novel binding site for VP35 described in this work (the CD), in addition to its other well-characterized N-terminal domain functions (aa 1-450) (29, 31, 32).…”

“…These results demonstrate that VP35 is necessary and sufficient for NP(1-641) localization to IBs. VP35 is a co-factor for EBOV RNA synthesis and also has anti-interferon (IFN) activity (31). Since VP35 fails to trigger IB formation on its own (10, 34), these data also demonstrate that in the context of NP(1-641) expression, only two proteins, NP and VP35, are sufficient for IB formation.…”

“…Given the important role of the NP 481-500 region in IB formation and interaction with VP35, and considering the known functions of NP and VP35 in viral RNA synthesis (31), we asked if the NP 481-500 region is important for reporter activity in p0 cells, which is directly dependent on viral transcription and RNA replication (54-56). Accordingly, trVLP assays of our three alanine-scanning mutants and the corresponding wild-type constructs were performed.…”

Identification of RUVBL1 and RUVBL2 as Novel Cellular Interactors of the Ebola Virus Nucleoprotein

“…Any ancillary roles of NP remain to be determined. Filovirus RNA synthesis occurs in cytoplasmic inclusions where PPIs among replication complex constituents NP, VP35, VP30, and L are well characterized [68,69,70]. Notably, expression of VP35, VP30, and L proteins individually results in diffuse localization patterns of each, whereas the individual expression of NP results in the formation of cytoplasmic inclusions that resemble the sites of replication during infection.…”

Crippling life support for SARS-CoV-2 and other viruses through synthetic lethality

“…These interactions can inhibit existing functions, redirect host proteins to other locations, or generate new targets and functionalities, which may lead to other downstream network effects. Because viruses depend on these host functions usurped by protein-protein interactions, targeting the interfaces between virus and host proteins directly has been proposed as a mechanism to inhibit the viral life cycle (Basler et al, 2019;Carpp et al, 2014;Gordon et al, 2020a;Heaton, 2019;Luo et al, 2016). However, we propose that the susceptible state induced by these interactions may be best targeted by chemically exploiting the genetic concept of synthetic lethality.…”