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.

FIG. 1.

JUNV RNA genome termini. (A) Virus RNA terminal sequences were experimentally determined by 3′ RACE and RNA ligation (Exp) and are shown in comparison with available sequences from GenBank (GB). Nucleotide differences are shown underlined, and g signifies an extra G found at the 5′ end of approximately 25 to 50% of the population of JUNV S RNAs analyzed. (B) Predicted panhandle structures for both S and L genomic RNAs.

FIG. 2.

(A) Schematic of plasmids indicating resulting primary transcripts. (B) Functional L protein required for minigenome replication. BSR-T7/5 cells growing in 24-well plates were transfected with pJunS-ΔGPC:GFP and pJunL (right) or pJunL-ΔSDD (left). Transfected cells were fixed 3 dpt, counterstained with DAPI and photographed with UV light in the GFP range (green cells). (C) Functional L protein required for virus replication and spread. BSR-T7/5 cells were transfected with pJunS and pJunL (right) or pJunL-ΔSDD (left). JUNV proteins were detected 5 dpt with anti-Junin rabbit serum and anti-rabbit Alexa Fluor 594 (red stain). Cells were counterstained with DAPI and photographed with UV light in a regular upright microscope.

FIG. 3.

(A) Schematic of plasmids encoding virus GPC cleavage variants. Plasmid primary transcript is shown 3′ to 5′, and the encoded GPC cleavage site is indicated for each plasmid. (B) Replication of wt and cleavage variant JUNV in transfected cells. BSR-T7/5 cells were transfected with pJunS, pJunS-ΔSKI or pJunS-Furin with pJunL or pJunL-ΔSDD. N protein was detected 3 dpt with anti-Junin rabbit serum and an anti-rabbit Alexa Fluor 594 (red stain). GPC was detected with a MAb anti-Junin GPC and anti-mouse Alexa Fluor 488 (green stain). Cells were counterstained with DAPI and photographed with UV light in a confocal microscope. (C) Virus GPC cleavage by SKI-1/S1P or furin-like PCs is required for rescue of infectious rJUNV. The efficiency of viral rescue was examined by focus formation in Vero E6 cells (1st and 2nd passages) and by plaque assay of virus stocks (2nd passage). +2 to + 5 indicates an average of 2 to 5 large (>50 cell) foci of infected cells/well on a six well plate.

FIG. 4.

rJUNV-Furin exhibits altered virus growth and glycoprotein maturation. (A) Vero E6 cells were infected with wt JUNV (triangle), rJUNV (square) and rJUNV-Furin viruses (circle). Duplicate samples were collected and titers determined at 0, 1, 3, 5, and 7 days p.i. (B) Protein lysates from Vero infected cells were examined by Western blot using anti-JUNV GPC MAbs. Glycosylated forms of GPC and G2 forms are indicated with open arrows and unglycosylated forms with black arrows. EndoH resistant forms of GPC (asterisk), and G2 (triangle and circle) are also indicated. Blot was reprobed with anti-actin MAb to control the amount of loaded proteins in the gel.

FIG. 5.

rJUNV-Furin grows in SKI-1/S1P deficient cells. SRD12B cells deficient in SKI-1/S1P were infected with rJUNV or rJUNV-Furin in the absence or presence of 30 μM the furin inhibitor dec-RVKR-CMK. Thirty-six hours p.i. supernatants were replaced with fresh media with or without inhibitor. JUNV proteins were detected 3 dpi using an anti-Junin rabbit serum and an anti-rabbit Alexa Fluor 546 (red stain). Cells were counterstained with DAPI and photographed with UV light in a regular upright microscope.