RNA processing bodies are disassembled during Old World alphavirus infection.

RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3-4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.

Highly Pathogenic New World Arenavirus Infection Activates the Pattern Recognition Receptor Protein Kinase R without Attenuating Virus Replication in Human Cells.

The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.

Density thresholds for Mopeia virus invasion and persistence in its host Mastomys natalensis.

Well-established theoretical models predict host density thresholds for invasion and persistence of parasites with a density-dependent transmission. Studying such thresholds in reality, however, is not obvious because it requires long-term data for several fluctuating populations of different size. We developed a spatially explicit and individual-based SEIR model of Mopeia virus in multimammate mice Mastomys natalensis. This is an interesting model system for studying abundance thresholds because the host is the most common African rodent, populations fluctuate considerably and the virus is closely related to Lassa virus but non-pathogenic to humans so can be studied safely in the field. The simulations show that, while host density clearly is important, sharp thresholds are only to be expected for persistence (and not for invasion), since at short time-spans (as during invasion), stochasticity is determining. Besides host density, also the spatial extent of the host population is important. We observe the repeated local occurrence of herd immunity, leading to a decrease in transmission of the virus, while even a limited amount of dispersal can have a strong influence in spreading and re-igniting the transmission. The model is most sensitive to the duration of the infectious stage, the size of the home range and the transmission coefficient, so these are important factors to determine experimentally in the future.

Meta-analysis of high-throughput datasets reveals cellular responses following hemorrhagic fever virus infection.

The continuing use of high-throughput assays to investigate cellular responses to infection is providing a large repository of information. Due to the large number of differentially expressed transcripts, often running into the thousands, the majority of these data have not been thoroughly investigated. Advances in techniques for the downstream analysis of high-throughput datasets are providing additional methods for the generation of additional hypotheses for further investigation. The large number of experimental observations, combined with databases that correlate particular genes and proteins with canonical pathways, functions and diseases, allows for the bioinformatic exploration of functional networks that may be implicated in replication or pathogenesis. Herein, we provide an example of how analysis of published high-throughput datasets of cellular responses to hemorrhagic fever virus infection can generate additional functional data. We describe enrichment of genes involved in metabolism, post-translational modification and cardiac damage; potential roles for specific transcription factors and a conserved involvement of a pathway based around cyclooxygenase-2. We believe that these types of analyses can provide virologists with additional hypotheses for continued investigation.

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

A role for the C terminus of Mopeia virus nucleoprotein in its incorporation into Z protein-induced virus-like particles.

Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.

Receptor binding and cell entry of Old World arenaviruses reveal novel aspects of virus-host interaction.

Ten years ago, the first cellular receptor for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the highly pathogenic Lassa virus (LASV) was identified as alpha-dystroglycan (alpha-DG), a versatile receptor for proteins of the extracellular matrix (ECM). Biochemical analysis of the interaction of alpha-DG with arenaviruses and ECM proteins revealed a strikingly similar mechanism of receptor recognition that critically depends on specific sugar modification on alpha-DG involving a novel class of putative glycosyltransferase, the LARGE proteins. Interestingly, recent genome-wide detection and characterization of positive selection in human populations revealed evidence for positive selection of a locus within the LARGE gene in populations from Western Africa, where LASV is endemic. While most enveloped viruses that enter the host cell in a pH-dependent manner use clathrin-mediated endocytosis, recent studies revealed that the Old World arenaviruses LCMV and LASV enter the host cell predominantly via a novel and unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the virus is rapidly delivered to endosomes via an unusual route of vesicular trafficking that is largely independent of the small GTPases Rab5 and Rab7. Since infection of cells with LCMV and LASV depends on DG, this unusual endocytotic pathway could be related to normal cellular trafficking of the DG complex. Alternatively, engagement of arenavirus particles may target DG for an endocytotic pathway not normally used in uninfected cells thereby inducing an entry route specifically tailored to the pathogen's needs.

Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses.

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.

Basolateral entry and release of New and Old World arenaviruses from human airway epithelia.

Transmission of arenaviruses from rodent hosts to humans is generally thought to occur through inhalation or ingestion of dust or droplets containing viral particles. Here we demonstrate that two identified arenavirus receptors, alpha-dystroglycan (alpha-DG) and transferrin receptor 1 (TfR1), are expressed in polarized human airway epithelia. Lymphocytic choriomeningitis virus strains with high or low alpha-DG affinity and Junin virus, which binds TfR1, efficiently infected polarized epithelia only when applied to the basolateral surface or when injury compromised tight junction integrity. Viral egress from infected epithelia exhibited basolateral polarity. This study demonstrates that respiratory entry of arenaviruses occurs via basolateral receptors.

Old World arenavirus infection interferes with the expression of functional alpha-dystroglycan in the host cell.

alpha-Dystroglycan (alpha-DG) is an important cellular receptor for extracellular matrix (ECM) proteins as well as the Old World arenaviruses lymphocytic choriomeningitis virus (LCMV) and the human pathogenic Lassa fever virus (LFV). Specific O-glycosylation of alpha-DG is critical for its function as receptor for ECM proteins and arenaviruses. Here, we investigated the impact of arenavirus infection on alpha-DG expression. Infection with an immunosuppressive LCMV isolate caused a marked reduction in expression of functional alpha-DG without affecting biosynthesis of DG core protein or global cell surface glycoprotein expression. The effect was caused by the viral glycoprotein (GP), and it critically depended on alpha-DG binding affinity and GP maturation. An equivalent effect was observed with LFVGP. Viral GP was found to associate with a complex between DG and the glycosyltransferase LARGE in the Golgi. Overexpression of LARGE restored functional alpha-DG expression in infected cells. We provide evidence that virus-induced down-modulation of functional alpha-DG perturbs DG-mediated assembly of laminin at the cell surface, affecting normal cell-matrix interactions.

Human macrophages, but not dendritic cells, are activated and produce alpha/beta interferons in response to Mopeia virus infection.

Lassa virus (LV) and Mopeia virus (MV) are closely related members of the Arenavirus genus, sharing 75% amino acid sequence identity. However, LV causes hemorrhagic fever in humans and nonhuman primates, whereas MV cannot induce disease. We have previously shown that antigen-presenting cells (APC)-macrophages (MP) and dendritic cells (DC)-sustain high replication rates of LV but are not activated, suggesting that they play a role in the immunosuppression observed in severe cases of Lassa fever. Here, we infected human APC with MV and analyzed the cellular responses induced. MV infection was productive in MP and even more so in DC. Apoptosis was not induced in either cell type. Moreover, unlike DC, MP were early and strongly activated in response to MV, as shown by the increased surface expression of CD86, CD80, CD54, CD40, and HLA-abc and by the production of mRNA encoding alpha interferon (IFN-alpha), IFN-beta, tumor necrosis factor alpha and interleukin-6. In addition, MV-infected MP produced less of the virus than DC, which was related to the fact that these cells secreted IFN-alpha. Thus, the strong activation of MP is probably a major event in the control of MV infection and may be involved in the induction of an adaptive immune response in infected hosts. These results may explain the difference in pathogenicity between LV and MV.

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor.

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.