Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease.
2019; 26 (4): 906
The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.
View details for PubMedID 30673613
A Viral Protein Restricts Drosophila RNAi Immunity by Regulating Argonaute Activity and Stability
CELL HOST & MICROBE
2018; 24 (4): 542-+
The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.
View details for PubMedID 30308158
Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein
JOURNAL OF VIROLOGY
2017; 91 (5)
Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.
View details for DOI 10.1128/JVI.01779-16
View details for Web of Science ID 000394356400008
View details for PubMedID 28003491
View details for PubMedCentralID PMC5309961
RNA Interference-Mediated Intrinsic Antiviral Immunity in Invertebrates
2013; 371: 183–200
In invertebrates such as insects and nematodes, RNA interference (RNAi) provides RNA-based protection against viruses. This form of immunity restricts viral replication and dissemination from infected cells and viruses, in turn, have evolved evasion mechanisms or RNAi suppressors to counteract host defenses. Recent advances indicate that, in addition to RNAi, other related small RNA pathways contribute to antiviral functions in invertebrates. This has led to a deeper understanding of fundamental aspects of small RNA-based antiviral immunity in invertebrates and its contribution to viral spread and pathogenesis.
View details for DOI 10.1007/978-3-642-37765-5_7
View details for Web of Science ID 000330591400008
View details for PubMedID 23686236
Slicer Activity in Drosophila melanogaster S2 Extract
ANTIVIRAL RNAI: CONCEPTS, METHODS, AND APPLICATIONS
2011; 721: 231–44
Reconstitution of RNA-inducing silencing complex (RISC) in vitro is a powerful biochemical technique to analyze crucial steps in RNA interference (RNAi) pathways. RISC contains an RNase enzyme, Argonaute, which is guided by small interfering RNA (siRNA) to recognize and silence its targets. Drosophila S2 cell extract is a good source of enzymes and factors that faithfully recapitulate essential steps in RISC assembly and function. In this chapter, we will describe how to prepare enzymatically active cell-free S2 extract to analyze the Slicer activity of RISC as well as the effects of viral RNAi suppressors which block this process.
View details for DOI 10.1007/978-1-61779-037-9_14
View details for Web of Science ID 000288921500014
View details for PubMedID 21431689
Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2010; 17 (5): 547–U41
Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.
View details for DOI 10.1038/nsmb.1810
View details for Web of Science ID 000277330700006
View details for PubMedID 20400949
View details for PubMedCentralID PMC3815677
Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication
JOURNAL OF VIROLOGY
2006; 80 (19): 9865–75
The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.
View details for DOI 10.1128/JVI.00561-06
View details for Web of Science ID 000240647200051
View details for PubMedID 16973591
View details for PubMedCentralID PMC1617274
Caliciviruses differ in their functional requirements for eIF4F components
JOURNAL OF BIOLOGICAL CHEMISTRY
2006; 281 (35): 25315–25
Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.
View details for DOI 10.1074/jbc.M602230200
View details for Web of Science ID 000240031300033
View details for PubMedID 16835235
Functional analyses of RNA structures shared between the internal ribosome entry sites of hepatitis C virus and the picornavirus porcine teschovirus 1 Talfan
JOURNAL OF VIROLOGY
2006; 80 (3): 1271–79
The internal ribosome entry site (IRES) of porcine teschovirus 1 (PTV-1), a member of the Picornaviridae family, is quite distinct from other well-characterized picornavirus IRES elements, but it displays functional similarities to the IRES from hepatitis C virus (HCV), a member of the Flaviviridae family. In particular, a dominant negative mutant form of eIF4A does not inhibit the activity of the PTV-1 IRES. Furthermore, there is a high level (ca. 50%) of identity between the PTV-1 and HCV IRES sequences. A secondary-structure model of the whole PTV-1 IRES has been derived which includes a pseudoknot. Validation of specific features within the model has been achieved by mutagenesis and functional assays. The differences and similarities between the PTV-1 and HCV IRES elements should assist in defining the critical features of this type of IRES.
View details for DOI 10.1128/JVI.80.3.1271-1279.2006
View details for Web of Science ID 000234871400021
View details for PubMedID 16415004
View details for PubMedCentralID PMC1346926
Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro
JOURNAL OF VIROLOGY
2005; 79 (12): 7698–7706
The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.
View details for DOI 10.1128/JVI.79.12.7698-7706.2005
View details for Web of Science ID 000229416100041
View details for PubMedID 15919922
View details for PubMedCentralID PMC1143669