Publications by Year: 2013

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis. In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications. Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses. The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines, which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

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.