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.     

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.     

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.     

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.     

Sodium Hydrogen Exchangers Contribute to Arenavirus Cell Entry

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a great public health concern in the regions in which they are endemic. Moreover, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The limited existing armamentarium to combat human-pathogenic arenaviruses underscores the importance of developing novel antiarenaviral drugs, a task that would be facilitated by the identification and characterization of virus-host cell factor interactions that contribute to the arenavirus life cycle. A genome-wide small interfering RNA (siRNA) screen identified sodium hydrogen exchanger 3 (NHE3) as required for efficient multiplication of LCMV in HeLa cells, but the mechanisms by which NHE activity contributed to the life cycle of LCMV remain unknown. Here we show that treatment with the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) resulted in a robust inhibition of LCMV multiplication in both rodent (BHK-21) and human (A549) cells. EIPA-mediated inhibition was due not to interference with virus RNA replication, gene expression, or budding but rather to a blockade of virus cell entry. EIPA also inhibited cell entry mediated by the glycoproteins of the HF arenaviruses LASV and Junin virus (JUNV). Pharmacological and genetic studies revealed that cell entry of LCMV in A549 cells depended on actin remodeling and Pak1, suggesting a macropinocytosis-like cell entry pathway. Finally, zoniporide, an NHE inhibitor being explored as a therapeutic agent to treat myocardial infarction, inhibited LCMV propagation in culture cells. Our findings indicate that targeting NHEs could be a novel strategy to combat human-pathogenic arenaviruses.     

Identification of a broad-spectrum arenavirus entry inhibitor

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.     

Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral trans-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.     

Pathogenic Old World arenaviruses inhibit TLR2/Mal-dependent proinflammatory cytokines in vitro

Lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), the causative agent of Lassa fever (LF), have extensive strain diversity and significant variations in pathogenicity for humans and experimental animals. The WE strain of LCMV (LCMV-WE), but not the Armstrong (Arm) strain, induces a fatal LF-like disease in rhesus macaques. We also demonstrated that LASV infection of human macrophages and endothelial cells resulted in reduced levels of proinflammatory cytokines. Here we have shown that cells infected with LASV or with LCMV-WE suppressed Toll-like receptor 2 (TLR2)-dependent proinflammatory cytokine responses. The persisting isolate LCMV clone 13 (CL13) also failed to stimulate interleukin-6 (IL-6) in macrophages. In contrast, nonpathogenic Mopeia virus, which is a genetic relative of LASV and LCMV-Arm induced robust responses that were TLR2/Mal dependent, required virus replication, and were enhanced by CD14. Superinfection experiments demonstrated that the WE strain of LCMV inhibited the Arm-mediated IL-8 response during the early stage of infection. In cells transfected with the NF-κB-luciferase reporter, infection with LCMV-Arm resulted in the induction of NF-κB, but cells infected with LCMV-WE and CL13 did not. These results suggest that pathogenic arenaviruses suppress NF-κB-mediated proinflammatory cytokine responses in infected cells.     

Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-beta) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.     

Role of DC-SIGN in Lassa Virus Entry into Human Dendritic Cells

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.     

Absence of CTL responses to early viral antigens facilitates viral persistence

CD8+ T cells are crucial for the control of intracellular pathogens such as viruses and some bacteria. Using lymphocytic choriomeningitis virus (LCMV) infection of mice--the prototypic arenavirus evolutionarily closely related to human Lassa fever and South American hemorrhagic fever viruses, we have shown previously that the kinetics of Ag presentation determine immunodominance of the LCMV-specific CTL response due to progressive exhaustion of LCMV nucleoprotein (NP)-specific CTL upon increasing viral load. In this study, we provide evidence that CTL against early LCMV NP-derived epitopes are more important in virus control than those against late glycoprotein-derived epitopes. We show that mice that are tolerant to all NP-derived T cell epitopes are severely compromised in their ability to control larger inocula of LCMV, supporting our hypothesis that CD8+ T cells specific for early viral Ags play a major role in acute virus control. Thus, the kinetics with which virus-derived T cell epitopes are presented has a strong impact on the efficacy of the antiviral immunity. This aspect should be taken into consideration for the development of vaccines.     

Reverse genetics generation of chimeric infectious Junin/Lassa virus is dependent on interaction of homologous glycoprotein stable signal peptide and G2 cytoplasmic domains

The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.     

The genomic rate of molecular adaptation of the human influenza A virus

Quantifying adaptive evolution at the genomic scale is an essential yet challenging aspect of evolutionary biology. Here, we develop a method that extends and generalizes previous approaches to estimate the rate of genomic adaptation in rapidly evolving populations and apply it to a large data set of complete human influenza A virus genome sequences. In accord with previous studies, we observe particularly high rates of adaptive evolution in domain 1 of the viral hemagglutinin (HA1). However, our novel approach also reveals previously unseen adaptation in other viral genes. Notably, we find that the rate of adaptation (per codon per year) is higher in surface residues of the viral neuraminidase than in HA1, indicating strong antibody-mediated selection on the former. We also observed high rates of adaptive evolution in several nonstructural proteins, which may relate to viral evasion of T-cell and innate immune responses. Furthermore, our analysis provides strong quantitative support for the hypothesis that human H1N1 influenza experiences weaker antigenic selection than H3N2. As well as shedding new light on the dynamics and determinants of positive Darwinian selection in influenza viruses, the approach introduced here is applicable to other pathogens for which densely sampled genome sequences are available, and hence is ideally suited to the interpretation of next-generation genome sequencing data.     

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins–a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion–the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.