Efficient Reverse Genetics Generation of Infectious Junin Viruses Differing in Glycoprotein Processing

Abstract

The New World arenaviruses, Junin, Machupo, Guanarito, Sabia, and Chapare, are associated with rapidly progressing severe hemorrhagic fever with a high rate of case fatality in various regions of South America. The threat of natural or deliberate outbreaks associated with these viruses makes the development of preventive or therapeutic measures important. Here we describe a Junin virus functional minigenome system and a reverse genetics system for production of infectious Junin virus. This robust, highly efficient system involves transfection of cells with only two plasmids which transcribe the virus S and L antigenomic RNAs. The utility of the system is demonstrated by generating Junin viruses which encode a glycoprotein precursor (GPC) containing the following: (i) the wild-type (SKI-1/S1P peptidase) cleavage site, (ii) no cleavage site, or (iii) a cleavage site where the SKI-1/S1P motif (RSLK) is replaced by a furin cleavage site (RRKR). In contrast to the wild-type virus, Junin virus lacking a GPC cleavage site replicated within successfully transfected cells but failed to yield infectious virus particles. This confirms observations with other arenaviruses suggesting that GPC cleavage is essential for arenavirus infectivity. In contrast, infectious Junin virus which encoded GPC cleaved by furin-like proteases was easily generated. The two-plasmid, high efficiency aspects of this Junin virus reverse genetics system show great promise for addressing important questions regarding arenavirus hemorrhagic fever disease and for development of precisely attenuated live arenavirus vaccines.

Rescue of a Recombinant Machupo Virus from Cloned cDNAs and In Vivo Characterization in Interferon (αβ/γ) Receptor Double Knockout Mice

“…Our new data suggested two mismatches at positions 6 and 8 in the same region (Fig. 1B), which is consistent with the sequences of other arenaviruses such as JUNV and LASV (42,45,46). The terminal 19 nucleotides at the 5= and 3= ends of L and S genomic RNAs are highly conserved among arenaviruses and play critical roles in viral RNA replication and transcription.…”

Lassa Virus Nucleoprotein Mutants Generated by Reverse Genetics Induce a Robust Type I Interferon Response in Human Dendritic Cells and Macrophages

“…While cloning the S segment was relatively straightforward, three steps were required for the L segment, due to strong secondary structures in the intergenic region (IGR), impairing plasmid stability (cloning strategy available upon request). Similar difficulties in the cloning of the Junin virus L segment were described previously (1). We confirmed the published AV strain sequence (18,48) and added the 19 nucleotides missing at the 3Ј and 5Ј extremities of the L segment (EMBL accession numbers FR832710 and FR832711).…”

Substitutions in the Glycoprotein (GP) of the Candid#1 Vaccine Strain of Junin Virus Increase Dependence on Human Transferrin Receptor 1 for Entry and Destabilize the Metastable Conformation of GP

“…We cloned the Cd1 genomic S strand from VeroE6 cells infected with a stock of Cd1 (from USAMRIID; kindly provided by Michael Buchmeier, University of California, Irvine, CA) and sequenced multiple GP clones. We compared the resulting sequences to those of the parental XJ strain (34) and the attenuated intermediates XJ13 and XJ44 (4,5,26) (Fig. 1A).…”