Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA

Abstract

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.

Fig. 1.

Sequence of the LCMV L-IGR and 5′ terminus and genome expression vectors. (A) Schematic of the bisegmented LCMV genome with its four known genes (arrows). Inverted writing indicates antisense polarity. (B) Sequence analysis of the L-segment 5′ end was carried out as described in Supporting Text. The first-strand cDNA (cDNA) was A-tailed at its 3′ end for amplification by PCR and subsequent sequence analysis. The deducted 5′ terminal template sequence of the L-segment genome and antigenome, respectively, and their nucleotide positions are indicated (−1 to 9). Note the 5′ nontemplated G at position −1. (C) Intrasegment base-pairing and panhandle formation of the L-segment 5′ and 3′ terminal nucleotides with positions indicated. (D) Comparison of the L-segment antigenome 5′ end with its template, the L-segment genome 3′ terminus. Nucleotide positions are indicated. (E) Schematic description of the genome expression vectors used in this study. Noncoding genetic tags are indicated by ∗, cis-acting elements are boxed, and ORFs are labeled and represented as arrows. Inverted writing indicates antisense polarity. pol-I-P, pol I promoter; pol-I-T, pol I terminator.

Fig. 2.

Recovery of genetically tagged Cl13 entirely from cDNA. (A and B) As outlined schematically in A, subconfluent BHK-21 cells in M6 tissue culture wells were transfected with 0.8 μg of pC-NP, 1 μg of pC-Cl13-L, 0.8 μg of pI-S-Cl13(−)∗, and either 1.4 μg of pI-L-Cl13(−) to recover rCl13∗ or 1.4 μg of pI-L-Cl13(−)∗ to recover rCl13∗∗. In negative control wells [without (w/o) pI-S-Cl13(−)∗], the pI-S-Cl13(−)∗ plasmid was substituted for an MG expression plasmid lacking LCMV structural genes and expressing GFP instead (9). (B) Culture supernatant was collected at the indicated time points and tested for viral infectivity by immunofocus assays. Symbols represent individual culture wells. (C) Schematic description of the genetic tags in the S- and L-segment cDNAs. The stretches of viral RNA amplified by RT-PCR and the predicted fragments obtained from restriction digestion are indicated. (D and E) Fresh BHK-21 cells were infected with wtCl13, rCl13∗, and rCl13∗∗ at multiplicity of infection of 0.1 or left uninfected (none). Forty-eight hours later, total cellular RNA was harvested and processed for RT-PCR amplification. L segment-specific (D) and S segment-specific (E) amplification products (+RT, Top) yielded the expected fragments upon digestion with AvaII or EcoNI, respectively (Middle), and were fully RT-dependent (compare with −RT, Bottom). Single-headed arrow: undigested PCR product; double-headed arrow: digested fragments; feathered arrow: primer dimers.

Fig. 3.

Propagation of cDNA-derived viruses in cell culture and infected mice. (A) Subconfluent BHK-21 cells in M6 tissue culture wells were infected with wtCl13, rCl13∗, or rCl13∗∗ at multiplicity of infection of 0.01. Supernatant was collected at the indicated time points for determination of viral titers. Symbols represent the mean ± SD of three individual culture wells. (B) C57BL/6 mice were infected with 2 × 10⁶ PFU of ARM53b, wtCl13, rCl13∗, or rCl13∗∗. Blood was collected at the indicated time points for the assessment of viral titers. Symbols represent the mean ± SD of four mice per group. One representative experiment of two is shown.

Fig. 4.

Suppression of B cell and T cell responses by the cDNA-derived viruses. On day 0, C57BL/6 mice (same mice as in Fig. 3B) were infected i.v. (1° infection) with 2 × 10⁶ PFU of ARM53b, wtCl13, rCl13∗, or rCl13∗∗, or they were left uninfected (none). Twenty-two days later, the indicated groups of mice were challenged with 2 × 10⁶ PFU of VSV i.p. (2° infection). (A) On days 25 and 29 (3 and 7 days after secondary infection), serum samples were collected for determination of VSV-neutralizing IgM (day 25) and neutralizing IgG (day 29). Symbols represent individual mice. One representative experiment of two is shown. (B) On day 30, the mice were killed, and the frequency of splenic IFN-γ-producing CD8⁺ T cells specific for either the immunodominant VSV NP-derived epitope NP52 or the LCMV NP-derived epitope NP396 was determined in an intracellular cytokine assay. Background IFN-γ production is shown for comparison (unstimulated). The frequency of IFN-γ-producing CD8⁺ cells within the total CD8⁺ T cell compartment is indicated for each representative FACS plot. Numbers indicate the mean ± SD of three mice per group.