Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature Drosophila C Virus (DCV) infection causes low mortality and persistent infection, whereas the closely related Cricket Paralysis Virus (CrPV) causes a lethal infection. We show these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA (dsRNA) and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Ago2 and inhibits its activity, without affecting the miRNA-Ago1 mediated silencing. The link between viral RNAi suppressors and the outcome of infection was examined using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a dramatic 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.

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.

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.Picornaviruses, including foot-and-mouth disease virus (FMDV), poliovirus (PV), and human rhinoviruses (HRVs), have a positive-sense RNA genome of about 8 kb that is infectious (1, 5). Following virus attachment and entry into the cell, the RNA genome is delivered into the cytoplasm and translation of the genome is required to produce the viral proteins that are necessary for virus assembly and RNA replication. The genome encodes a single large polyprotein that is processed, largely by internal trans-acting proteases, to produce about 12 mature proteins plus various precursors (some of these have distinct functions). The proteins encoded within the P1 region form the capsid, while proteins encoded in the P2 and P3 regions are required for RNA replication. After some rounds of translation, there has to be a switch so that translation of the viral RNA is stopped and RNA replication can commence, since these two processes appear incompatible on the same RNA molecule (11). Most picornaviruses replicate with high efficiency within susceptible cells, and within a few hours, the amount of viral RNA can represent 5% of the total RNA in cells (a level similar to that of all the cellular cytoplasmic mRNAs together). Nearly all of the FMDV RNA generated by replication is infectious; indeed, microinjection of cells with as little as 1 to 2 molecules of viral RNA is sufficient to initiate an infection (4). Thus, the replication of FMDV genomes within cells is remarkably efficient.To replicate the positive-sense genome, an antisense RNA has to be synthesized which then functions as the template for the production of new positive-sense infectious genomes (32). RNA is synthesized by the viral 3D protein that functions as an RNA-dependent RNA polymerase and will be referred to as 3D pol . Interestingly, 3D pol requires the uridylylated form of the 3B/VPg peptide (VPgpU or VPgpUpU) to act as the primer for both positive-and negative-strand synthesis. In recent...

SUMMARY The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and micro-cephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis pro-vide effective antivirals. We show that different cyto-solic 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 hu-man 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, mor-tality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.

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.Porcine teschovirus 1 (PTV-1) Talfan is the prototype member of the Teschovirus genus within the Picornaviridae family. PTV infection results in polioencephalomyelitis in swine, and there have been recent incidents of disease (resulting in paralysis or mortality) in both the United States and Japan (28,42). Multiple serotypes of teschoviruses have been identified, and nearly complete genome sequences are available for a variety of strains (6,14,20,43). The 5Ј-terminal region of the genome sequence is missing in each case, and it is possible that a poly(C) tract is present within the 5Ј untranslated region of the viral RNA in at least some strains (6, 43).Picornavirus genomes are infectious and function as mRNAs. Initiation of protein synthesis on picornavirus RNA is dependent on an internal ribosome entry site (IRES) (see reference 2 for a review). Several different classes of picornavirus IRES element have been described. With the exception of the PTV-1 IRES (see below), they are all large, complex RNA structures of about 450 nucleotides (nt) which contain a polypyrimidine tract located about 20 nt upstream of an AUG codon at the 3Ј end of the element. (Note that in the cardio-and aphthovirus elements, this AUG is an initiation codon, but in the enteroand rhinoviruses, the AUG codon is not usually recognized and initiation occurs at the next AUG codon.) The poliovirus (PV) and human rhinovirus elements represent one class of IRES; these elements function poorly in the rabbit reticulocyte lysate (RRL) in vitro translation system unless it is supplemented with additional proteins (e.g., from HeLa cell extracts). In contrast, the cardio-and aphthovirus IRES elements (e.g., from encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus [FMDV]) do function very efficiently in the standard RRL translation system and they have a secondary structure different from that of the entero-and rhinovirus IRES elements. The hepatitis A virus IRES is distinct again; most notably, it requires the intact translation initiation complex eIF4F (1, 3), whereas the other picornavirus IRES elements function when the eIF4G component of this complex has been cleaved or the cap-binding pro...

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 (3D pol ), 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.

Summary 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.

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.