Reverse genetics approaches to combat pathogenic arenaviruses

Abstract

Several arenaviruses cause hemorrhagic fever (HF) in humans, and evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Moreover, arenaviruses pose a biodefense threat. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with anemia and other side effects. Therefore, it is important to develop effective vaccines and better antiviral drugs to combat the dual threats of naturally occurring and intentionally introduced arenavirus infections. The development of arenavirus reverse genetic systems is allowing investigators to conduct a detailed molecular characterization of the viral cis-acting signals and trans-acting factors that control each of the steps of the arenavirus life cycle, including RNA synthesis, packaging and budding. Knowledge derived from these studies is uncovering potential novel targets for therapeutic intervention, as well as facilitating the establishment of assays to identify and characterize candidate antiviral drugs capable of interfering with specific steps of the virus life cycle. Likewise, the ability to generate predetermined specific mutations within the arenavirus genome and analyze their phenotypic expression would significantly contribute to the elucidation of arenavirus-host interactions, including the basis of their ability to cause severe HF. This, in turn, could lead to the development of novel, potent and safe arenavirus vaccines.

Fig. 1

LCMV genome organization.

Fig. 2

Basic aspects of arenavirus RNA replication and gene transcription illustrated for the S segment. The L polymerase associated with the virus RNP initiates transcription from the genome promoter located at the genome 3′-end. Primary transcription results in synthesis of NP mRNA (or L mRNA in the case of the L segment). Subsequently the virus polymerase can adopt a replicase mode and moves across of the IGR to generate a copy of the full-length antigenome (ag) S RNA (or ag L RNA). This ag S RNA will serve as template for the synthesis of the GP mRNA (the ag L RNA will serve as template for the synthesis of the Z mRNA). The agRNA species serve also as templates for the amplification of the corresponding genome RNA species.

Fig. 3

Schema of the LCMV MG rescue system. LCMV RNA analogues, or MG, with correct termini are synthesized intracellularly using either a T7 RP, or RNA pol-I, expression system. Cotransfection with plasmids expressing the viral trans-acting factors L and NP allow for the generation of viral RNP (MG RNA encapsidated by NP) that is substrate for the plasmid-supplied virus L polymerase. The virus polymerase directs the synthesis of two RNA species, the aMG RNA (replicative species) and a subgenomic mRNA that directs expression of the reporter gene of choice, CAT in this case. Incorporation of plasmids expressing Z and GP results in assembly and budding of infectious LCMV virus like particles (VLP).

Fig. 4

Rescue of infectious rLCMV entirely from cloned cDNAs. Cells are transfected with plasmids that directed T7RP-or pol-I mediated intracellular synthesis of L and S antigenomic (Lag and Sag) RNA species, together with pol-II expression plasmids for the viral trans-acting factors L and NP (and also T7RP if required). At different times after transfection, cell culture supernatants are tested for production of infectious rlCMV. Production of rLCMV is readily and consistently detected at 48 h after transfection, which is followed by a rapid increase in virus production reaching titers of 10⁷ PFU/ml between 60 and 72 h post-transfection. Notably, we observed similar rescue efficiencies using either genomic or antigenomic polarities of the L and S RNAs. This finding indicates that annealing between viral mRNAs and genome, or antigenome, RNA species do not pose a significant problem for the rescue of LCMV, and likely arenaviruses in general. Likewise, both the T7RP and pol-I based rescue systems exhibited similar efficiencies.

Fig. 5

Use of a tri-segments genome strategy to incorporate additional genes within the arenavirus genome. Each of the two S segments was altered to replace one of the viral ORF by the ORF of a gene of interest (GOI). The physical separation of GP and NP into two different S segments would represent a strong selective pressure to select and maintain a virus capable of packaging 1 L and 2 S segments.