Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production.

RIG-I is a major cytoplasmic sensor of viral pathogen-associated molecular pattern (PAMP) RNA and induces type I interferon (IFN) production upon viral infection. A double-stranded RNA (dsRNA)-binding protein, PACT, plays an important role in potentiating RIG-I function. We have shown previously that arenaviral nucleoproteins (NPs) suppress type I IFN production via their RNase activity to degrade PAMP RNA. We report here that NPs of arenaviruses block the PACT-induced enhancement of RIG-I function to mediate type I IFN production and that this inhibition is dependent on the RNase function of NPs, which is different from that of a known mechanism of other viral proteins to abolish the interaction between PACT and RIG-I. To understand the biological roles of PACT and RIG-I in authentic arenavirus infection, we analyze growth kinetics of recombinant Pichinde virus (PICV), a prototypical arenavirus, in RIG-I knockout (KO) and PACT KO mouse embryonic fibroblast (MEF) cells. Wild-type (WT) PICV grew at higher titers in both KO MEF lines than in normal MEFs, suggesting the important roles of these cellular proteins in restricting virus replication. PICV carrying the NP RNase catalytically inactive mutation could not grow in normal MEFs but could replicate to some extent in both KO MEF lines. The level of virus growth was inversely correlated with the amount of type I IFNs produced. These results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication and that viral NP RNase activity is essential for optimal viral replication by suppressing PACT-induced RIG-I activation.IMPORTANCE We report here a new role of the nucleoproteins of arenaviruses that can block type I IFN production via their specific inhibition of the cellular protein sensors of virus infection (RIG-I and PACT). Our results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication. This new knowledge can be exploited for the development of novel antiviral treatments and/or vaccines against some arenaviruses that can cause severe and lethal hemorrhagic fever diseases in humans.

In vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions.

UNLABELLED: Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCE: Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

The Z proteins of pathogenic but not nonpathogenic arenaviruses inhibit RIG-I-like receptor-dependent interferon production.

UNLABELLED: Arenavirus pathogens cause a wide spectrum of diseases in humans ranging from central nervous system disease to lethal hemorrhagic fevers with few treatment options. The reason why some arenaviruses can cause severe human diseases while others cannot is unknown. We find that the Z proteins of all known pathogenic arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Lassa, Junin, Machupo, Sabia, Guanarito, Chapare, Dandenong, and Lujo viruses, can inhibit retinoic acid-inducible gene 1 (RIG-i) and Melanoma Differentiation-Associated protein 5 (MDA5), in sharp contrast to those of 14 other nonpathogenic arenaviruses. Inhibition of the RIG-i-like receptors (RLRs) by pathogenic Z proteins is mediated by the protein-protein interactions of Z and RLRs, which lead to the disruption of the interactions between RLRs and mitochondrial antiviral signaling (MAVS). The Z-RLR interactive interfaces are located within the N-terminal domain (NTD) of the Z protein and the N-terminal CARD domains of RLRs. Swapping of the LCMV Z NTD into the nonpathogenic Pichinde virus (PICV) genome does not affect virus growth in Vero cells but significantly inhibits the type I interferon (IFN) responses and increases viral replication in human primary macrophages. In summary, our results show for the first time an innate immune-system-suppressive mechanism shared by the diverse pathogenic arenaviruses and thus shed important light on the pathogenic mechanism of human arenavirus pathogens. IMPORTANCE: We show that all known human-pathogenic arenaviruses share an innate immune suppression mechanism that is based on viral Z protein-mediated RLR inhibition. Our report offers important insights into the potential mechanism of arenavirus pathogenesis, provides a convenient way to evaluate the pathogenic potential of known and/or emerging arenaviruses, and reveals a novel target for the development of broad-spectrum therapies to treat this group of diverse pathogens. More broadly, our report provides a better understanding of the mechanisms of viral immune suppression and host-pathogen interactions.

Characterization of the host response to pichinde virus infection in the Syrian golden hamster by species-specific kinome analysis.

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens.

Inhibition of arenavirus infection by a glycoprotein-derived peptide with a novel mechanism.

UNLABELLED: The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 µM, with complete inhibition of viral plaque formation at approximately 20 µM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE: Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic and prone to treatment failure, we identified a peptide, AVP-p, derived from the fusion glycoprotein of a nonpathogenic model arenavirus, which demonstrates antiviral activity and no acute cytotoxicity. AVP-p is unique among self-derived inhibitory peptides in that it shows broad, specific activity against pseudoviruses bearing Old and New World arenavirus glycoproteins but not against viruses from other families. Further, the peptide's mechanism of action is highly novel. Biochemical assays and cryo-electron microscopy indicate that AVP-p induces premature activation of viral fusion proteins through membrane perturbance. Peptide treatment, however, does not increase the infectivity of cell-bound virus. We hypothesize that prematurely activated virions are less fit for receptor binding and membrane fusion and that AVP-p may represent a viable therapeutic strategy for arenavirus infection.

Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus.

We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.

Biological roles and functional mechanisms of arenavirus Z protein in viral replication.

Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

Proteomic analysis of Pichindé virus infection identifies differential expression of prothymosin-alpha.

The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-alpha. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.

Attenuated and lethal variants of Pichindé virus induce differential patterns of NF-kappaB activation suggesting a potential target for novel therapeutics.

Lassa virus pathogenesis is believed to involve dysregulation of cytokines. We have previously shown nuclear factor-kappaB (NF-kappaB) inhibition using a BSL-2 model for Lassa fever. Here we further define the potential mechanism for NF-kappaB inhibition as involving increased levels of repressive p50/p50 homodimers, and suggest a novel therapeutic strategy that acts via modulation of host signaling.

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus.

A virulent (P18) strain of the Pichinde arenavirus produces a disease in guinea pigs that somewhat mimics human Lassa fever, whereas an avirulent (P2) strain of this virus is attenuated in infected animals. It has been speculated that the composition of viral genomes may confer the degree of virulence in an infected host; the complete sequence of the viral genomes, however, is not known. Here, we provide for the first time genomic sequences of the S and L segments for both the P2 and P18 strains. Sequence comparisons identify three mutations in the GP1 subunit of the viral glycoprotein, one in the nucleoprotein NP, and five in the viral RNA polymerase L protein. These mutations, alone or in combination, may contribute to the acquired virulence of Pichinde virus infection in animals. The three amino acid changes in the variable region of the GP1 glycoprotein subunit may affect viral entry by altering its receptor-binding activity. While NP has previously been shown to modulate host immune responses to viral infection, we found that the R374 K change in this protein does not affect the NP function of suppressing interferon-beta expression. Four out of the five amino acid changes in the L protein occur in a small region of the protein that may contribute to viral virulence by enhancing its function in viral genomic RNA synthesis.

Arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism.

Arenaviruses are important causes of viral hemorrhagic fevers in humans. Arenavirus infection of cells occurs via a pH-dependent endocytic route, but detailed studies of entry pathways have not been done. We investigated the role of cell membrane cholesterol, caveolae, and clathrin coated pits in infection by Lassa virus (LASV), which utilizes alpha-dystroglycan (alpha-DG) as a receptor, and Pichindé virus (PICV), which does not. Depletion of cellular cholesterol by treatment with methyl betacyclodextrin (MbetaCD) or nystatin/progesterone inhibited PICV replication and transfer of packaged marker gene by LASV or PICV pseudotyped retroviral particles. In cells lacking caveolae due to silencing of the caveolin-1 gene, no inhibition of PICV infection or LASV pseudotype transduction was observed. However, PICV infection and LASV and PICV pseudotype transduction was inhibited when an Eps15 dominant negative mutant was used to inhibit clathrin-mediated endocytosis. Altogether, the results indicate that diverse arenaviruses have a common requirement for cell membrane cholesterol and clathrin mediated endocytosis in establishing infection.

Interferon alfacon-1 protects hamsters from lethal pichinde virus infection.

Hemorrhagic fever of arenaviral origin is a frequently fatal infectious disease of considerable priority to the biodefense mission. Historically, the treatment of arenaviral infections with alpha interferons has not yielded favorable results. Here we present evidence that interferon alfacon-1, a nonnaturally occurring bioengineered alpha interferon approved for the treatment of chronic hepatitis C, is active against Pichinde and Tacaribe arenaviruses in cell culture. In the hamster model of Pichinde virus (PCV) infection, interferon alfacon-1 treatment significantly protected animals from death, prolonged the survival of those that eventually died, reduced virus titers, and limited liver damage characteristic of PCV-induced disease. Moreover, interferon alfacon-1 also demonstrated therapeutic activity, to a lesser degree, when the initiation of treatment was delayed up to 2 days post-virus challenge. Despite the observed advantages of interferon alfacon-1 therapy, efforts to stimulate the immune system with the known interferon inducer poly(I:C12U) (Ampligen) offered only limited protection against lethal PCV challenge. Taken together, these data suggest that the increased potency of the bio-optimized interferon alfacon-1 molecule may be critical to the observed antiviral effects. These data are the first report demonstrating efficacious treatment of acute arenaviral disease with alpha interferon therapy, and further study is warranted.

Alterations in NF-kappaB and RBP-Jkappa by arenavirus infection of macrophages in vitro and in vivo.

Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-kappaB and RBP-Jkappa, we compared the activities of NF-kappaB and RBP-Jkappa in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-kappaB activation and caused a shift in the size of the RBP-Jkappa complex. The virulent P18 virus showed less inhibition of NF-kappaB and failed to alter the size of the RBP-Jkappa complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-kappaB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jkappa. By contrast, P18 induced large amounts of the NF-kappaB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jkappa size. Taken together, these changes suggest that the attenuated viral strain induces an "activation" of macrophages, while the virulent form of the virus does not.

Analysis of Pichinde arenavirus transcription and replication in human THP-1 monocytic cells.

Human promonocytic THP-1 cells were previously shown to be nonpermissive for Pichinde virus (PV) replication unless the cells were induced to differentiate to macrophages by stimulation with phorbol ester (PMA) (J. Virol. 65, 3575, 1991). The restriction did not involve receptor modulation, virus binding, nor internalization of virus but a requirement for a host cell function in PV replication was observed in that the phorbol ester effect required protein kinase C activation and was inhibited by actinomycin D. In this report we demonstrate that PV S RNA genomes, antigenomes, GPC mRNA and NP mRNA are expressed at high levels in PMA treated THP-1 cells but at significantly lower levels or not at all in untreated cells. We have also determined that degradation of input viral S RNA does not account for decreased PV RNA synthesis in the undifferentiated cells. This suggests that the restriction of PV replication in THP-1 cells is a post-penetration event which precedes transcription of viral mRNAs and replication of viral genomes and supports a role for differentiation-specific host cell factors early in PV replication.

The nucleoprotein of Pichinde virus expressed by a vaccinia-Pichinde virus recombinant partially protects hamsters from lethal virus challenge.

Syrian hamsters, strain MHA/Lak, are susceptible to intraperitoneal infection with Pichinde virus and die from an overwhelming viremia. We have studied the ability of a vaccinia-Pichinde recombinant virus expressing amino acids 51-561 of the viral nucleoprotein (VVNP51-561) to protect from lethal Pichinde virus infection. Priming with VVNP51-561 significantly delayed mortality and increased final survival outcome after challenge with 2 x 10(3) pfu of Pichinde virus. This protection was not complete compared to priming with Pichinde virus in the footpad, which was not lethal and provided 100% protection. At a higher challenge dose of Pichinde virus, 2 x 10(4) pfu, immunization with VVNP51-561 delayed mortality but did not increase final survival. The partial protection correlated with an early but not late reduction in infectious virus in serum, kidney and liver, and infectious centers in the spleen. Thus the immune response generated by VVNP51-561 could initially control the infection, effectively reducing the virus inoculum. As the infection proceeded, virus replication could not be limited resulting in death in some hamsters. The partial protection did not appear to be mediated by anti-viral antibodies since these were not detected in the serum of VVNP56-561-immunized hamsters. This finding appears to support the hypothesis that in many arenavirus infections cellular immunity is central to viral clearance and protection from reinfection.