Different Mechanisms of Cell Entry by Human-Pathogenic Old World and New World Arenaviruses

Abstract

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication

“…Recombinant wild-type LCMV (rLCMV/WT) and rLCM viruses expressing vesicular stomatitis virus G (rLCMV/VSV-G), or LASV GPC (rLCMV/LASV-GPC), instead of LCMV GPC, as well as the recombinant WT VSV (VSV/WT) and rVSV expressing GPC of LCMV instead of VSV G (rVSV/LCMV-GPC), has been previously described (17,21,23,24,28). To generate rLCMV GFP-P2A-NP, we replace the NP open reading frame (ORF) in plasmid moPol-I/Sag (17,21) by the ORF GFP-P2A-NP that contained the ORF of GFP tagged to the N terminus of NP, separated by the 2A peptide sequence derived from porcine teschovirus (PVT1: P2A) (27).…”

“…These advances in arenavirus molecular genetics have opened new avenues for the development of screening strategies to rapidly identify inhibitors of arenavirus multiplication. Furthermore, we have developed cell-based assays to examine individually each step of the arenavirus life cycle, which facilitates target identification and elucidation of mechanism of action of newly identified inhibitors of arenavirus multiplication (20)(21)(22)(23)(24)(25)(26). We document here the generation of a novel recombinant LCMV (rLCMV/GFP-P2A-NP) that expresses green fluorescent protein (GFP) from a bicistronic NP mRNA via the use of the small 2A peptide sequence (27) and its usage to establish a cell-based assay amenable to HTS for the identification of compounds with anti-LCMV activity.…”

Targeting the Proteolytic Processing of the Viral Glycoprotein Precursor Is a Promising Novel Antiviral Strategy against Arenaviruses

“…HEK293 cells were transfected with SuperFect (Qiagen) (38), and CHOK1, FD11, and SRD12B cells were transfected using Lipofectamine (17). Transfection efficiencies as determined by IF detection of transgenes were Ͼ90% for HEK293 cells and Ͼ60% for CHOK1, FD11, and SRD12B cells.…”

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

“…Moreover, upon M␤CD treatment, membrane cholesterol levels remain low for 9 to 12 h (25,43). Cells were treated with M␤CD (10 mM) for 1 h either 1 h prior to or 1 h after infection with BDV (MOI, 0.1), and at 48 h p.i., we examined the number of BDV-infected cells based on expression of BDV N as determined by IFA (Fig.…”