Promyelocytic leukemia protein is a restriction factor for Junín virus independently of Z matrix protein.

The New World (NW) mammarenavirus Junín (JUNV) is the etiological agent of Argentine hemorrhagic fever, a human endemic disease of Argentina. Promyelocytic leukemia protein (PML) has been reported as a restriction factor for several viruses although the mechanism/s behind PML-mediated antiviral effect may be diverse and are a matter of debate. Previous studies have reported a nuclear to cytoplasm translocation of PML during the murine Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) infection. This translocation was found to be mediated by the viral Z protein. Here, we show that PML restricts JUNV infection in human A549 cells. However, in contrast to LCVM, JUNV infection enhances PML expression and PML is not translocated to the cytoplasm neither it colocalizes with JUNV Z protein. Our study demonstrates that a NW mammarenavirus as JUNV interacts differently with the antiviral protein PML than LCMV.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor.

UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.

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.

Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections.

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.

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.

Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.

Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.

Glial fibrillary acidic protein (GFAP) immunochemical profile after junin virus infection of rat cultured astrocytes.

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining (P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

Galectin-3 is upregulated in activated glia during Junin virus-induced murine encephalitis.

Argentine haemorrhagic fever (AHF) is a systemic febrile syndrome characterized by several haematological and neurological alterations caused by Junín virus (JUNV), a member of the Arenaviridae family. Newborn mice are highly susceptible to JUNV and the course of infection has been associated with the viral strain used. Galectin-3 (Gal-3) is an animal lectin that has been proposed to play an important role in some central nervous system (CNS) diseases. In this study, we analysed Gal-3 expression at the transcriptional and translational expression levels during JUNV-induced CNS disease. We found that Candid 1 strain induced, with relatively low mortality, a subacute/chronic CNS disease with significant glia activation and upregulation of Gal-3 in microglia cells as well as in reactive astrocytes that correlated with viral levels. Our results suggest an important role for Gal-3 in viral-induced CNS disease.

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro.

In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.

Influence of volume expansion on capillary transport in the gut of Pichindé virus infected strain 13 guinea pigs.

Hydropenic strain 13 guinea pigs (GP13) injected with Pichindé virus show marked jejunal capillary disruption determined by a reduction in protein reflection coefficient (sigma) [Katz and Starr, 1990]. These studies examined whether this is reversed by a 2% Ringer's expansion, and whether anorexia may contribute to reduced sigma. GP13 were either infected or given a 60% expected ad lib diet, and were studied while under a 2% Ringer's expansion. The results showed 1) infected GP13 separated into groups with either a persistently low sigma (volume unresponsive) or a normal sigma (volume responsive), but no obvious feature of the animals could predict responsiveness; 2) caloric restriction had minimal to no effect on sigma; and 3) both responsive and unresponsive GP had increased heart, lung, and kidney weights. Volume expansion may have salutary effects in some infected GP13, and this is probably due to pressure mediated effects on the capillary wall. The findings support the possibility that conservative management of hemorrhagic fevers may directly ameliorate capillary leak.

Pichinde virus infection in strain 13 guniea pigs reduces intestinal protein reflection coefficient with compensation.

Pichinde virus inoculation into strain 13 guinea pigs is a model with features reputed to be similar to hemorrhagic fever in humans. Although the infection is lethal by day 13-19, guinea pigs of approximately 600 g do not show edema or effusions. This raises the questions of whether capillary damage is present in such infected animals and, if it is, why edema is absent. The effects of Pichinide virus on protein transport across jejunal capillaries were examined in 38 normal and 7 infected strain 13 guinea pigs 12 days after inoculation. The latter lost 20.3% body weight but maintained normal blood pressure, serum protein concentration, and jejunal lymph flow. However, their protein solvent drag reflection coefficient (sigma) was reduced to .52 +/- .03 (mean +/- SE) from .73 +/- .02 (2P less than .001), while permeability-surface area product was not changed. In the absence of gross edema or effusions, Pichinde virus-infected guinea pigs demonstrated a leaky gut capillary wall to protein compatible with an increase in pore size or large pore number less than sufficient to change permeability-surface area product. Compensatory mechanisms that prevent edema at this stage are efficient and may include reduced capillary pressure or some degree of capillary flow stasis.

Changes in platelet-activating factor, catecholamine, and serotonin concentrations in brain, cerebrospinal fluid, and plasma of pichinde virus-infected guinea pigs.

Brain concentrations of platelet-activating factor (PAF), catecholamines, and serotonin were measured in control and Pichinde virus-infected strain 13 guinea pigs on postinoculation day (PID) 12. After virus inoculation, PAF concentrations increased 81% in cerebrum, 147% in diencephalon-brain stem, and 110% in cerebellum from baseline values of 2.6 +/- 0.3, 4.3 +/- 0.2, and 6.1 +/- 0.5 (ng/g wet tissue), respectively. Dopamine concentrations in the infected cerebrum and diencephalon-brain stem increased significantly, whereas norepinephrine concentration increased only in cerebrum. However, serotonin concentrations in all three regions of infected brain decreased significantly as compared with control values. There were no significant changes in epinephrine concentrations of infected brain. Norepinephrine and epinephrine concentrations in plasma and cerebrospinal fluid on PID 7 and 12 increased significantly as compared with control values, while plasma dopamine concentration increased significantly on PID 7. Increased brain PAF, dopamine, and and norepinephrine concentrations with decreased brain serotonin concentrations may mediate the hyperactivity of the hypothalamic-pituitary-adrenal axis and involve some unknown pathophysiologic processes of arenaviral infection. Furthermore, increased plasma catecholamine concentrations are associated with stress and may be partially responsible for the development of cardiovascular dysfunction and pulmonary edema during this viral disease.

Role of atrial natriuretic peptide in disturbed water and electrolyte metabolism of guinea pigs infected with Pichinde virus.

Daily intake and output of water and electrolytes (Na+, K+, and Cl-) were determined for 14 days in control and Pichinde virus-infected strain-13 guinea pigs. Although water intake began to decrease 7 days after virus inoculation, total daily water output (insensible water loss, urine excretion, plus fecal water loss) had little change. However, insensible water loss alone increased markedly in the virus-infected animals. Both intake and excretions of urinary and fecal electrolytes decreased at the middle (days 7 to 10) and late (days 11 to 14) stages of viral infection. Differences between intake and output of water and electrolytes were reduced significantly during these periods. To determine a possible relationship between atrial natriuretic peptide (ANP) and urinary Na+ and water excretion over intake, we measured plasma ANP concentrations. The mean control value of plasma ANP was 24 +/- 1.0 pg/ml, and plasma ANP concentrations of infected animals increased significantly (P < 0.01) to 49.5 +/- 3.9 and 51.3 +/- 8.8 pg/ml on postinoculation days 7 and 12. Because the overall physiologic responses to Pichinde virus infection are complicated, it is difficult to postulate a single central theme concerning the pathogenesis. Nevertheless, we hypothesize that the virus-induced invisible tissue "biochemical lesions" and the consequential release of mediators and hormones were possible causes of death. Among other pathophysiologic mechanisms, the increased plasma ANP concentration may have played a role in the development of disturbed water and electrolytes metabolism during Pichinde virus infection.

Effect of interferon on togavirus and arenavirus infections of animals.

The sensitivity of togaviruses to the effects of IF appears to permit successful exploitation of IF inducers, whereas the relative insensitivity of arenaviruses to IF in vitro appears to correlate with the complete lack of success of poly (ICLC) therapy in the monkey model. Noteworthy is the successful use of poly (ICLC) against fatal encephalitis caused by JE virus in monkeys. Additional studies appear to be warranted to characterize further the role of IF and/or IF inducers in other model infections prior to studies in man.

Astrocyte differentiation induced by Junín virus in rat brain cell cultures.

Morphological and immunocytochemical differentiation was observed in astroglial cell cultures of the rat infected with Junín virus. From days 3 to 6 postinoculation (p.i.), GFAP immunostaining was observed in both the perikaryon and processes of maturated astrocytes, whereas it was limited to the perikaryon in less differentiated cells. The rather slow spontaneous differentiation usually occurring in astroglial cell cultures was seen to be accelerated by viral infection, mimicking the astrocytic reaction formerly described in Junín virus-inoculated mice. Infected cell monolayers showed orderly development, maintenance of contact inhibition, and exhaustion of cell cultures beyond the 6th-7th passages. The morphological and immunocytochemical maturation effects of Junín virus on astroglial cells were evident, but to a significantly lesser degree than those caused by rat brain extract. The glial cell cultures proved a valuable tool for the study of virus-cell interaction, since the immune response and the structural complexity of the whole animal can thus be avoided.