Tissue Tropism and Target Cells of NSs-Deleted Rift Valley Fever Virus in Live Immunodeficient Mice

Rift Valley fever, caused by a member of the Bunyaviridae family, has spread during recent years to most sub-Saharan African countries, in Egypt and in the Arabian peninsula. The virus can be transmitted by insect vectors or by direct contacts with infectious tissues. The analysis of virus replication and dissemination in laboratory animals has been hampered by the need to euthanize sufficient numbers of animals and to assay appropriate organs at various time points after infection to evaluate the viral replication. By following the bioluminescence and fluorescence of Rift Valley fever viruses expressing light reporters, we were able to track the real-time dissemination of the viruses in live immunodeficient mice. We showed that the first infected organs were the thymus, spleen and liver, but the liver rapidly became the main location of viral replication. Phagocytes also appeared as important targets, and their systemic depletion by use of clodronate liposomes decreased the number of viruses in the blood, delayed the viral dissemination and prolonged the survival of the infected mice

Mutational analysis of Rift Valley fever phlebovirus nucleocapsid protein indicates novel conserved, functional amino acids

Rift Valley fever phlebovirus (RVFV; Phenuiviridae, Phlebovirus) is an important mosquito-borne pathogen of both humans and ruminants. The RVFV genome is composed of tripartite, single stranded, negative or ambisense RNAs. The small (S) segment encodes both the nucleocapsid protein (N) and the non-structural protein (NSs). The N protein is responsible for the formation of the viral ribonucleoprotein (RNP) complexes, which are essential in the virus life cycle and for the transcription and replication of the viral genome. There is currently limited knowledge surrounding the roles of the RVFV nucleocapsid protein in viral infection other than its key functions: N protein multimerisation, encapsidation of the RNA genome and interactions with the RNA-dependent RNA polymerase, L. By bioinformatic comparison of the N sequences of fourteen phleboviruses, mutational analysis, minigenome assays and packaging assays, we have further characterised the RVFV N protein. Amino acids P11 and F149 in RVFV N play an essential role in the function of RNPs and are neither associated with N protein multimerisation nor known nucleocapsid protein functions and may have additional roles in the virus life cycle. Amino acid Y30 exhibited increased minigenome activity despite reduced RNA binding capacity. Additionally, we have determined that the N-terminal arm of N protein is not involved in N-L interactions. Elucidating the fundamental processes that involve the nucleocapsid protein will add to our understanding of this important viral protein and may influence future studies in the development of novel antiviral strategies

A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning

International audienceThe bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure