Inhibition of the PI3K/Akt pathway by Ly294002 does not prevent establishment of persistent Junín virus infection in Vero cells.

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

Altered expression of cytokines in mice infected intranasally with two syncytial variants of Herpes simplex virus type 1.

Immune evasion strategies are important for the onset and the maintenance of viral infections. Many viruses have evolved mechanisms to counteract or suppress the host immune response. We have previously characterized two syncytial (syn) variants of Herpes simplex 1 (HSV-1) strain F, syn14-1 and syn17-2, obtained by selective pressure with a natural carrageenan. These variants showed a differential pathology in vaginal and respiratory mucosa infection in comparison with parental strain. In this paper, we evaluated the modulation of immune response in respiratory mucosa by these HSV-1 variants. We observed altered levels of Tumor Necrosis Factor-α and Interleukin-6 in lungs of animals infected with the syn14-1 and syn17-2 variants compared with the parental strain. Also, we detected differences in the recruitment of immune cells to the lung in syn variants infected mice. Both variants exhibit one point mutation in the sequence of the gene of glycoprotein D detected in the ectodomain of syn14-1 and the cytoplasmic tail of syn17-2. Results obtained in the present study contribute to the characterization of HSV-1 syn variants and the participation of the cellular inflammatory response in viral pathogenesis.

Junin virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2α phosphorylation.

Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.

Differential effect of acute and persistent Junin virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B.

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.

Emergence of herpes simplex virus-1 syncytial variants with altered virulence for mice after selection with a natural carrageenan.

BACKGROUND: Antiviral therapy against herpes simplex virus based on sulfated polysaccharides, like carrageenans, represents a new alternative for genital herpes infections treatment and arises the concern about the appearance of resistant viral populations. METHODS: We characterized the F strain of herpes simplex virus-1 passaged in the presence of a natural carrageenan isolated from the red seaweed Gigartina skottbergii in view of the virulence for mice of isolated viral clones. RESULTS: Viral clones (syn14-1 and syn17-2) showed a syncytial phenotype and a mild resistance to carrageenan, heparin, acyclovir, and brivudine. Both clones were avirulent for BALB/c mice when inoculated intravaginally, whereas F strain produced high mortality. Attenuation correlated with low levels of TNF-[alpha], interleukin-6, and IFN-[gamma] in vaginal lavages although virus titers were similar to those obtained for F strain. On the contrary, they showed a marked virulence when inoculated intranasally leading to a generalized spreading of virus. CONCLUSIONS: Results confirm the hypothesis that selection of herpes simplex virus-1 with a carrageenan in vitro leads to the emergence of variants with a differential virulence when compared to the original virus. This finding should be addressed when an antiviral therapy against genital herpes infection employing a natural carrageenan is under consideration.

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.

Superinfection exclusion in BHK-21 cells persistently infected with Junín virus.

We characterized a persistently Junín virus (JUNV)-infected BHK-21 cell line obtained by experimental infection with the XJCl3 strain. This cell line, named K3, produced low levels of virus in supernatants which were not influenced by the presence of defective interfering (DI) particles after the first year of infection. K3 cells were able to exclude superinfection of the homologous JUNV and the antigenically related Tacaribe virus (TCRV), whereas the non-related arenaviruses lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV) could replicate normally. Although superinfecting virus binding and internalization to persistently infected cells were slightly reduced, earlier biosynthesis of antigenomic RNA was observed in comparison with BHK-21 cells. Despite the fact that superinfection did not increase the number of cells expressing viral antigens, de novo synthesis of superinfecting virus proteins was detected. The virus produced by JUNV-superinfected K3 cells remained mostly cell-associated in the form of particles tethered to the plasma membrane and aberrant tubular structures. JUNV restriction was correlated with an overexpression of cellular protein TSG101 in K3 cells, which has been pointed out as involved in the budding of several RNA viruses. This correlation was also observed in a cell clone isolated from K3. Reduction of TSG101 expression favoured the release of infectious virus to the supernatant of JUNV-superinfected K3 cells. Our data suggest that overexpression of TSG101 in K3 cells is a novel mechanism that may contribute, along with a diminished synthesis of superinfecting virus proteins, to explain superinfection exclusion in persistently arenavirus-infected cells.