Role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and RNA replication

Abstract

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has a bisegmented negative-strand RNA genome. Each segment carries two viral genes in opposite orientation and separated by an intergenic region (IGR). The RNA-dependent RNA polymerase (RdRp) L of LCMV produces subgenomic mRNA and full-length genomic and antigenomic RNA species in two different processes termed transcription and replication, respectively. It is widely accepted that intracellular nucleoprotein (NP) levels regulate these two processes. Intracellular NP levels increase during the course of the infection, resulting in the unfolding of secondary RNA structures within the IGR. Structure-dependent transcription termination at the IGR is thereby attenuated, promoting replication of genome and antigenome RNA species. To test this hypothesis, we established a helper-virus-free minigenome (MG) system where intracellular synthesis of an S segment analogue from a plasmid is driven by RNA polymerase I. Cotransfection with two additional plasmids expressing the minimal viral trans-acting factors L and NP under control of RNA polymerase II allowed for RNA synthesis mediated by the intracellularly reconstituted LCMV polymerase. Both processes, transcription and replication, were strictly dependent on NP. However, both were equally enhanced by incrementally increasing amounts of NP up to levels in the range of those in LCMV-infected cells. Our data are consistent with a central role for NP in transcription and replication of the LCMV genome, but they do not support the participation of NP levels in balancing the two processes.

FIG. 1.

Schematic of the pol-I-driven LCMV MG, showing also transcription and replication intermediates. pMG-ARM/S contains the elements described for the MG-ARM/S construct (14) flanked by the murine pol-I promoter (pol Ip) and terminator sequences (pol It) of pRF42 (9). Transcription of pMG-ARM/S by the cellular pol-I (primary transcription) generates MG-ARM/S RNA (MG), detectable by a CAT sense riboprobe. Replication of the MG (solid right arrow) yields an aMG RNA, detectable by a CAT antisense riboprobe. This aMG serves as a template for synthesis of more MG RNA by the virus polymerase (replication [Rep]; solid left arrow). Both MG and aMG RNA species are assumed to have a nontemplated G at their 5′ ends (12). Transcription (Tx; dashed arrow) of the MG RNA by the virus polymerase yields a subgenomic-length CAT mRNA that terminates within the IGR and that is detectable by a CAT antisense riboprobe. Subgenomic mRNA species are assumed to have 5′ end cap structures, containing 4 or 5 nt not derived from viral template sequences (XXXX) (20). cis-Acting sequences are in bold. IGR, intergenic region; Gr and Nr, sequences within the GP and NP ORFs, respectively.

FIG. 2.

Subcellular distribution of plasmid-supplied LCMV MG RNA and derived RNA species and MG-derived CAT activity. BHK-21 cells in six-well plates (80% confluent) were transfected with pMG-ARM/S (0.5 μg), pC-NP (0.8 μg), and pC-L (1 μg) in the combinations indicated at the bottom by using Lipofectamine as described previously (23). At 72 h posttransfection cells were harvested. For each sample, one-half of the cells were used to prepare nuclear and cytoplasmic RNA as described previously (6). Ten percent of the total cytoplasmic (lanes 1 to 5) or nuclear (lanes 6 to 10) RNA obtained from each sample was analyzed on duplicate blots by Northern hybridization, thereby normalizing for the amount of RNA on a per-cell basis. (A) Ethidium bromide staining of 28S rRNA showed comparable total RNA amounts loaded for samples 1 to 5 and 6 to 10. (B) Hybridization to a CAT sense riboprobe. (C) Hybridization to a CAT antisense riboprobe. (D) The other half of each cell sample was processed for CAT assay as described previously (5). O, origin of sample application; NAc, nonacetylated chloramphenicol; MAc, monoacetylated chloramphenicol; DAc, diacetylated chloramphenicol.

FIG. 3.

Influence of intracellular NP levels on transcription and replication of the LCMV MG. BHK-21 cells in six-well plates (80% confluent) were transfected with pMG-ARM/S (0.5 μg), pC-L (0.2 μg), the amount of pC-NP indicated, and empty pC to normalize the total amount of plasmid DNA transfected in each case to 2.3 μg. Cells were harvested 40 h posttransfection. For each sample 50% of the cells were used for isolation of total cellular RNA and the other 50% were used to determine NP expression levels by Western blotting. (A) Analysis of RNA synthesis mediated by the LCMV polymerase. Equal amounts of RNA of each sample, as determined by ethidium bromide (Eth Br) staining of the 28S rRNA, were analyzed by Northern blot hybridization with a CAT antisense probe to detect CAT mRNA and aMG RNA species. After hybridization with the CAT antisense (AS) probe and phosphorimager analysis of the blot, the membrane was stripped and hybridized with a probe to the housekeeping gene encoding GAPDH to confirm that the membrane contained similar amounts of RNA in each lane. One representative experiment of six is shown. (B) The intensities of aMG and CAT mRNA bands shown in panel A were assessed by phosphorimager. The hybridization signal obtained in the absence of NP (A, lane 1) was considered nonspecific and subtracted from all the samples. We observed similar background hybridization signals in the absence of L (A, lane 9). Phosphorimager values for each sample were normalized with respect to the corresponding GAPDH hybridization signals. Normalized signals were depicted as percentages of replication (aMG RNA) or transcription (CAT mRNA) with respect to the values (100%) obtained with 0.4 μg of pC-NP. The replicate (aMG)-to-transcript (CAT mRNA) ratio for each sample (bottom) was calculated based on the normalized phosphorimager values obtained for the corresponding bands shown in panel A. One representative experiment of six is shown. (C) Expression levels of NP protein in transfected cells. Equal protein amounts for each sample, as determined by Coomassie blue staining (i), were analyzed by Western blotting using a gp polyclonal serum against LCMV that recognizes the virus NP (ii). The membrane was stripped and probed with rabbit serum against actin to verify that the membrane contained similar protein amounts in each lane (iii). (D) Expression levels of NP protein in LCMV-infected cells. BHK-21 cells were infected with LCMV (multiplicity of infection, 1), and at the indicated times after infections cell extracts were prepared for analysis of protein expression by Western blotting. Equal amounts of protein were loaded for each sample and analyzed for NP expression levels as described for panel C. One representative experiment of two is shown. p.i., postinfection.

FIG. 4.

Influence of intracellular NP levels on CAT activity. BHK-21 cells were seeded and transfected as described for Fig. 3A and B. Total cell extracts were processed for CAT assay as described previously (5). (A) Autoradiography of the thin-layer chromatography plate. Abbreviations: O, origin of sample application; NAc, nonacetylated chloramphenicol; MAc, monoacetylated chloramphenicol. No double-acetylated form of chloramphenicol was generated. (B) Chloramphenicol conversion was quantitated by phosphorimager analysis.