Junín Virus Promotes Autophagy To Facilitate the Virus Life Cycle.

Junín virus (JUNV), a member of the family Arenaviridae, is the etiological agent of Argentine hemorrhagic fever (AHF), a potentially deadly endemic-epidemic disease affecting the population of the most fertile farming land of Argentina. Autophagy is a degradative process with a crucial antiviral role; however, several viruses subvert the pathway to their benefit. We determined the role of autophagy in JUNV-infected cells by analyzing LC3, a cytoplasmic protein (LC3-I) that becomes vesicle membrane associated (LC3-II) upon induction of autophagy. Cells overexpressing enhanced green fluorescent protein (EGFP)-LC3 and infected with JUNV showed an increased number of LC3 punctate structures, similar to those obtained after starvation or bafilomycin A1 treatment, which leads to autophagosome induction or accumulation, respectively. We also monitored the conversion of LC3-I to LC3-II, observing LC3-II levels in JUNV-infected cells similar to those observed in starved cells. Additionally, we kinetically studied the number of LC3 dots after JUNV infection and found that the virus activated the pathway as early as 2 h postinfection (p.i.), whereas the UV-inactivated virus did not induce the pathway. Cells subjected to starvation or pretreated with rapamycin, a pharmacological autophagy inductor, enhanced virus yield. Also, we assayed the replication capacity of JUNV in Atg5 knockout or Beclin 1 knockdown cells (both critical components of the autophagic pathway) and found a significant decrease in JUNV replication. Taken together, our results constitute the first study indicating that JUNV infection induces an autophagic response, which is functionally required by the virus for efficient propagation.IMPORTANCE Mammalian arenaviruses are zoonotic viruses that cause asymptomatic and persistent infections in their rodent hosts but may produce severe and lethal hemorrhagic fevers in humans. Currently, there are neither effective therapeutic options nor effective vaccines for viral hemorrhagic fevers caused by human-pathogenic arenaviruses, except the vaccine Candid no. 1 against Argentine hemorrhagic fever (AHF), licensed for human use in areas of endemicity in Argentina. Since arenaviruses remain a severe threat to global public health, more in-depth knowledge of their replication mechanisms would improve our ability to fight these viruses. Autophagy is a lysosomal degradative pathway involved in maintaining cellular homeostasis, representing powerful anti-infective machinery. We show, for the first time for a member of the family Arenaviridae, a proviral role of autophagy in JUNV infection, providing new knowledge in the field of host-virus interaction. Therefore, modulation of virus-induced autophagy could be used as a strategy to block arenavirus infections.

Human transferrin receptor triggers an alternative Tacaribe virus internalization pathway.

Tacaribe virus (TCRV) entry occurs by receptor-mediated endocytosis. To explore the entry mechanism used by TCRV, the inhibitory effects of drugs and dominant negative (DN) constructions affecting the main endocytic pathways were analyzed. In cells lacking the human transferrin receptor (hTfR), compounds and DN proteins that impair clathrin-mediated endocytosis were shown to reduce virus internalization without affecting virion binding. In contrast, in cells expressing the hTfR, compounds that affect clathrin-mediated endocytosis did not affect TCRV infection. Destabilization of cholesterol-rich plasma membrane microdomains by treatment with nystatin was not able to block virus entry in the presence of hTfR. However methyl-ß-cyclodextrin, which extracts cholesterol from cell membranes, reduced virus internalization in cells expressing the hTfR. Inhibition of dynamin and neutralization of the pH of intracellular vesicles reduced virus internalization in all cell lines tested. Taken together, these results demonstrate that in cells expressing the hTfR, TCRV internalization depends on the presence of cholesterol, dynamin and acidic intracellular vesicles, while in the rest of the cell lines analyzed, clathrin-mediated endocytosis is the main TCRV entry pathway and, as expected, depends on dynamin and acidic intracellular vesicles. These results represent an important contribution to the characterization of the arenavirus replication cycle.

Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes.

Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-ß-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.

Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus, Junín virus.

The target cell tropism of enveloped viruses is regulated by interactions between viral proteins and cellular receptors determining susceptibility at a host cell, tissue or species level. However, a number of additional cell-surface moieties can also bind viral envelope glycoproteins and could act as capture receptors, serving as attachment factors to concentrate virus particles on the cell surface, or to disseminate the virus infection to target organs or susceptible cells within the host. Here, we used Junín virus (JUNV) or JUNV glycoprotein complex (GPC)-pseudotyped particles to study their ability to be internalized by the human C-type lectins hDC- or hL-SIGN. Our results provide evidence that hDC- and hL-SIGN can mediate the entry of Junín virus into cells, and may play an important role in virus infection and dissemination in the host.

An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection.

It has been previously described that S-layer binds to the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209). It was also shown that DC-SIGN is a cell-surface adhesion factor that enhances viral entry of several virus families. Among those, Junin virus (JUNV) entry is enhanced in cells expressing DC-SIGN and for that reason surface-layer protein (S-layer) of Lactobacillus acidophilus ATCC 4365 was evaluated as a possible JUNV inhibitor. Experiments using 3T3 cells stably expressing DC-SIGN, showed an almost complete inhibition of JUNV infection when they were treated with S-layer in a similar extend as the inhibition shown by mannan. However no inhibition effect was observed in 3T3 wild type cells or in 3T3 cells expressing liver/lymph node-specific ICAM-3 grabbing nonintegrin (L-SIGN or DC-SIGNR or CD209L). Treatments with S-layer during different times in the infection demonstrated that inhibition was only observed when S-layer was presented in early stages of the viral infection. This inhibition does not involve the classic recognition of mannose by this C-type lectin as the S-layer showed no evidence to be glycosylated. In fact, the highly basic nature of the S-layer (pI>9.5) seems to be involved in electrostatic interactions between DC-SIGN and S-layer, since high pH abolished the inhibitory effect on infection cause by the S-layer. In silico analysis predicts a Ca(2+)-dependant carbohydrate recognition domain in the SlpA protein. This novel characteristic of the S-layer, a GRAS status protein, contribute to the pathogen exclusion reported for this probiotic strain and may be applied as an antiviral agent to inhibit several kinds of viruses.

Involvement of cytoskeleton in Junín virus entry.

The early events in Junín virus (JUNV) infection are not thoroughly understood. We have previously shown that JUNV enter cells by clathrin-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early virus infection. Inhibitory effects of drugs affecting main cytoskeletal components on JUNV entry into Vero cells were analyzed. Drugs that disrupted microfilaments or stabilized microtubules inhibited early steps of virus entry. In contrast, drugs that stabilized microfilaments or depolymerized microtubules were not able to block virus entry very efficiently. Furthermore, real time PCR was performed to detect viral entry and we found more than 10-fold less RNA when microfilaments were depolymerized while a 100-fold diminution was seen when microtubules were stabilized. Taken together our results demonstrate that JUNV relies on an intact actin network during early infection in Vero cells while a dynamic microtubule network is also needed. This represents an important contribution to the characterization of arenavirus multiplication cycle.

Entry Studies of New World Arenaviruses.

Identification of cell moieties involved in viral binding and internalization is essential since their expression would render a cell susceptible. Further steps that allow the uncoating of the viral particle at the right subcellular localization have been intensively studied. These "entry" steps could determine cell permissiveness and often define tissue and host tropism. Therefore applying the right and, when possible, straightforward experimental approaches would shorten avenues to the complete knowledge of this first and key step of any viral life cycle. Mammarenaviruses are enveloped viruses that enter the host cell via receptor-mediated endocytosis. In this chapter we present a set of customized experimental approaches and tools that were used to describe the entry of Junín virus (JUNV), and other New World mammarenavirus members, into mammalian cells.

Intermediate filament integrity is required for Junin virus replication.

The role of the cytoskeletal framework in Junin virus (JUNV) replication has already been demonstrated with compounds interfering with the microfilament (MF) and microtubule (MT) networks. In this work, we evaluated the role of intermediate filaments (IF) during JUNV infection. We tested the effect of acrylamide, a compound that selectively disrupts IF, in culture of three different cell types: Vero cells, murine astrocytes and human foreskin fibroblasts. Perturbation of intermediate filaments had an inhibitory effect on JUNV production within a range of acrylamide concentration of 0.5-3mM in a dose-dependent manner, without cell viability modification. Recovery experiments showed that viral production was partially increased when medium containing acrylamide was replaced by normal maintenance medium (MM). The adsorption and internalization steps were not affected by IF disruption. The expression of JUNV proteins was highly reduced in the presence of 2mM acrylamide while immunofluorescence staining of IF showed network disruption with the formation of cytoplasmic aggregates containing vimentin or glial fibrillary acidic protein (GFAP). We conclude that the IF network may play a role in the early step of JUNV multiplication, subsequent to virus entry and that its integrity is a necessary condition for the normal replication of JUNV in neural and fibroblast cells as well as in the Vero cell line.

Differential inhibitory action of two azoic compounds against arenaviruses.

The action of five azo-based compounds against the arenaviruses Junin (JUNV) and Tacaribe (TCRV) was evaluated in vitro by a virus yield inhibition assay in Vero cells and a cell-free virion inactivation assay. The compound 2-azo-(1'-(2'-nitroso)naphthyl)-benzoate (ANNB) was the most effective inhibitor of arenavirus production in Vero cells with EC(50) (effective concentration 50%) values in the range 6.5-26.2 microM and without inactivating properties. By contrast, the azodicarbonamide (ADA) was very effective in inactivating both arenaviruses with IC(50) (inactivating concentration 50%) values of 7.6 and 5.3 microM against JUNV and TCRV, respectively. The virucidal activity of ADA was time- and temperature-dependent. ANNB had no inhibitory action on virus binding or penetration of target cells and did not affect the synthesis of viral proteins. The most likely event susceptible to ANNB would be the process of intracellular virion assembly.

Involvement of cellular proteins in Junin arenavirus entry.

Junin arenavirus (JUNV) entry is dependent on clathrin-mediated pathways and it relies on the integrity of the actin cytoskeleton as well as the dynamics of microtubules. To determine the method of entry used by this human pathogen, we have demonstrated that in Vero cells JUNV is trafficked via the cellular dynamin 2 (dyn2) endocytic pathway and it is dependent on the Eps15 GTPase. In addition, we have shown that the virus travels through Rab5-mediated early and Rab7-mediated late endosomes in its pH-dependent entry. Altogether, this study gives further inside into the endocytic pathway utilized by the arenavirus JUNV.

Characterization of Junin arenavirus cell entry.

Junín virus (JUNV) entry is conducted by receptor-mediated endocytosis. To explore the cellular entry mechanism of JUNV, inhibitory effects of drugs affecting the main endocytic pathways on JUNV entry into Vero cells were analysed. Compounds that impair clathrin-mediated endocytosis were shown to reduce virus internalization without affecting virion binding. In contrast, drugs that alter lipid-raft microdomains, impairing caveola-mediated endocytosis, were not able to block virus entry. To show direct evidence of JUNV entry, transmission electron microscopy was performed; it showed JUNV particles of about 60-100 nm in membrane depressions that had an electron-dense coating. In addition, JUNV particles were found within invaginations of the plasma membrane and vesicles that resembled those of pits and clathrin-coated vesicles. Taken together, these results demonstrate that clathrin-mediated endocytosis is the main JUNV entry pathway into Vero cells and represent an important contribution to the characterization of the arenavirus multiplication cycle.

Polarized entry and release of Junin virus, a New World arenavirus.

Junin virus (JUNV), the causative agent of Argentine haemorrhagic fever, is a human pathogen that naturally enters the body through the epithelial cells of the respiratory and digestive tracts. The interaction of JUNV with two types of polarized epithelial cultures, Vero C1008 and A549, was investigated. Radioactive virus-binding assays showed that JUNV infects polarized lines preferentially through the apical surface. High-level expression of viral nucleoprotein was detected in polarized cell lines infected through the apical domain. Virus production from apical media was about 100-fold higher than that found into the basolateral medium. Confocal-immunofluorescence analysis revealed high-level expression of glycoprotein at the apical-membrane surface. Disruption of the microtubule network by colchicine impaired JUNV vectorial release. This is the first study to analyse the interaction between a member of the virus family Arenaviridae and polarized epithelial cells, showing preferential entry and release from the apical plasma membrane.

Susceptibilidad del ratón de 11 días a la infección con virus Junin propagado en distintos huéspedes / Susceptibility of 11 days old mice to infection with Junin virus propagated in several different hosts

Se analizaron algunas propiedades de doce clones de virus Junín obtenidos en células Vero, comparándolos entre sí, y respecto del "stock" parental preparado en cerebro de ratón lactante. No se encontraron diferencias significativas entre los mismos y en relación al virus parental cuando se determinaron la termolabilidad, termosensibilidad y patogenicidad para ratón lactante de 2 días de edad. En cambio el índice de virulencia (UFP/DL) para el ratón de 11 días resultó entre 50 y 100 veces menor para los clones analizados. Se pudo demostrar que esta variación en la virulencia era dependiente del huésped donde multiplicó en virus ya que un solo pasaje del "stock" parental por células Vero o BHK redujo 100 a 1000 veces el índice de virulencia para el ratón de 11 días, mientras que un pasaje posterior por cerebro de ratón, revirtió el índice a valores semejantes al obtenido para el "stock" de cerebro. Significativamente cuando el virus de cerebro sufrió un pasaje por células de embrión de ratón originó una población de virulencia intermedia. La dependencia de la virulencia con el huésped se obtuvo en forma independiente de la cepa de virus utilizada y solo se manifestó entre los 10 y 12 días de edad, antes y después de ese período las curvas de mortalidad de los ratones siguieron patrones similares. Estos resultados sugieren que la presencia de antígenos histocompatibles desempeñan un rol principal en la producción de la encefalitis, lo que se manifiesta claramente a la edad de 11 días