Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis

AbstractEbola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30–eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30–eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.</jats:p

Aptamers in Virology: Recent Advances and Challenges

Aptamers generated from randomized libraries of nucleic acids have found utility in a wide variety of fields and in the clinic. Aptamers can be used to target both intracellular and extracellular components, including small molecules, proteins, cells, and viruses. With recent technological developments in stringent selection and rapid isolation strategies, it is likely that aptamers will continue to make an impact as useful tools and reagents. Although many recently developed aptamers are intended for use as therapeutic and diagnostic agents, use of aptamers for basic research, including target validation, remains an active area with high potential to impact our understanding of molecular mechanisms and for drug discovery. In this brief review, we will discuss recent aptamer discoveries, their potential role in structural virology, as well as challenges and future prospects

VP24-Karyopherin alpha binding affinities differ between Ebolavirus species, nfluencing interferon inhibition and VP24 stability

Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCE The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis

Ebola virus VP35 interaction with dynein LC8 regulates viral RNA synthesis

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8

Nipah virus V protein binding alters MDA5 helicase folding dynamics

Nipah virus (NiV) is an emerging and deadly zoonotic paramyxovirus that is responsible for periodic epidemics of acute respiratory illness and encephalitis in humans. Previous studies have shown that the NiV V protein antagonizes host antiviral immunity, but the molecular mechanism is incompletely understood. To address this gap, we biochemically characterized NiV V binding to the host pattern recognition receptor MDA5. We find that the C-terminal domain of NiV V (

Cryo-EM analysis of Ebola virus nucleocapsid-like assembly

This protocol describes the reconstitution of the filamentous Ebola virus nucleocapsid-like assembl

The cap-snatching SFTSV endonuclease domain is an antiviral target

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (I

Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication

CAPG is required for Ebola virus infection by controlling virus egress from infected cells

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells