Cybele C. García

Inhibition of Junín virus replication by small interfering RNAs.

Abstract Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179–197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.

Host cell factors as antiviral targets in arenavirus infection.

Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.

An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein.

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication

We have recently demonstrated that AR‐12 (OSU‐03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR‐12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR‐12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α—dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over‐expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR‐12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR‐12—stimulated the co‐localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug‐induced autophagosome formation and reduced the anti‐viral protection afforded by AR‐12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR‐12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR‐12 acts as a broad‐specificity anti‐viral drug in vitro and in vivo. We argue future patient studies with AR‐12 are warranted. J. Cell. Physiol. 231: 2286–2302, 2016.

Cellular Promyelocytic Leukemia Protein Is an Important Dengue Virus Restriction Factor

The intrinsic antiviral defense is based on cellular restriction factors that are constitutively expressed and, thus, active even before a pathogen enters the cell. The promyelocytic leukemia (PML) nuclear bodies (NBs) are discrete nuclear foci that contain several cellular proteins involved in intrinsic antiviral responses against a number of viruses. Accumulating reports have shown the importance of PML as a DNA virus restriction factor and how these pathogens evade this antiviral activity. However, very little information is available regarding the antiviral role of PML against RNA viruses. Dengue virus (DENV) is an RNA emerging mosquito-borne human pathogen affecting millions of individuals each year by causing severe and potentially fatal syndromes. Since no licensed antiviral drug against DENV infection is currently available, it is of great importance to understand the factors mediating intrinsic immunity that may lead to the development of new pharmacological agents that can boost their potency and thereby lead to treatments for this viral disease. In the present study, we investigated the in vitro antiviral role of PML in DENV-2 A549 infected cells.

Progress for Antiviral Development in Latin America

This chapter summarizes the main contributions for antiviral development from scientists in Latin America in the last decade. Antiviral research in this region has been mainly focused on the search for alternative therapeutic agents for herpes simplex virus infections and the development of antiviral strategies to handle neglected viral diseases that represent a serious threat for local public health. The antiviral potential of natural extracts and compounds isolated from a vast diversity of plants, algae, and other natural sources has also been explored. Similarly, the study of the efficacy and mode of action of a variety of synthetic compounds reveals the ongoing progress for antiviral research in the region. In addition, approaches based on host factors as antiviral targets and the use of bioinformatics methods that allow structure-based design of viral inhibitors have strongly emerged during the last years as powerful tools used by Latin-American researchers for the development of novel antiviral strategies.

Determining the Virus Life-Cycle Stage Blocked by an Antiviral

Among the members of the Arenaviridae family, Junín virus and Lassa virus represent important human health threats generating annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of Argentina and Western Africa, respectively. Given the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. During the last two decades, academic research studies originated important results identifying novel molecules to be considered for further in vivo characterization. This chapter summarizes experimental in vitro approaches used to determine the possible mechanism of action of these antiviral agents.

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET‐UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non‐cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose‐dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6 h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT‐PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host‐targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses.