Publications

Despite countermeasures against influenza virus that prevent (vaccines) and treat (antivirals) infection, this upper respiratory tract human pathogen remains a global health burden, causing both seasonal epidemics and occasional pandemics. More potent and safe new vaccine technologies would contribute significantly to the battle against influenza and other respiratory infections. Using plasmid-based reverse genetics techniques, we have developed a single-cycle infectious influenza virus (sciIV) with immunoprotective potential. In our sciIV approach, the fourth viral segment, which codes for the receptor-binding and fusion protein hemagglutinin (HA), has been removed. Thus, upon infection of normal cells, although no infectious progeny are produced, the expression of other viral proteins occurs and is immunogenic. Consequently, sciIV is protective against influenza homologous and heterologous viral challenges in a mouse model. Vaccination with sciIV protects in a dose- and replication-dependent manner, which is attributed to both humoral responses and T cells. Safety, immunogenicity, and protection conferred by sciIV vaccination were also demonstrated in ferrets, where this immunization additionally blocked direct and aerosol transmission events. All together, our studies suggest that sciIV may have potential as a broadly protective vaccine against influenza virus.

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae(1), is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA(2-5). Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Arenaviruses have a bisegmented, negative-strand RNA genome. Both the large (L) and small (S) genome segments use an ambisense coding strategy to direct the synthesis of two viral proteins. The L segment encodes the virus polymerase (L protein) and the matrix Z protein, whereas the S segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC). NPs are the most abundant viral protein in infected cells and virions and encapsidate genomic RNA species to form an NP-RNA complex that, together with the virus L polymerase, forms the virus ribonucleoprotein (RNP) core capable of directing both replication and transcription of the viral genome. RNP formation predicts a self-association property of NPs. Here we document self-association (homotypic interaction) of the NP of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), as well as those of the hemorrhagic fever (HF) arenaviruses Lassa virus (LASV) and Machupo virus (MACV). We also show heterotypic interaction between NPs from both closely (LCMV and LASV) and distantly (LCMV and MACV) genetically related arenaviruses. LCMV NP self-association was dependent on the presence of single-stranded RNA and mediated by an N-terminal region of the NP that did not overlap with the previously described C-terminal NP domain involved in either counteracting the host type I interferon response or interacting with LCMV Z.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

Arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. Currently, there are no FDA-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. Arenavirus nucleoprotein (NP) is found associated with the genomic RNA forming the viral ribonucleoproteins (vRNPs) that together with the polymerase (L) direct viral replication and transcription. Virion formation requires the recruitment of vRNPs into budding sites, a process in which the arenavirus matrix-like protein (Z) plays a major role. Therefore, proper NP-NP and NP-Z interactions are required for the generation of infectious progeny. In this work we demonstrate the role of the amino acid residue D471 in the self-association of lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP). Amino acid substitutions at this position abrogate NP oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to dsRNA analogs was retained. Additionally, we also document the dominant negative effect of D471G mutation on viral infection, suggesting that NP self-association is an excellent target for the development of new antivirals against arenaviruses.

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.