Molecular cloning and host range analysis of three cytomegaloviruses from Mastomys natalensis

Abstract

Herpesvirus-based vectors are attractive for use as conventional or transmissible vaccines against emerging zoonoses in inaccessible animal populations. In both cases, cytomegaloviruses (CMVs) as members of the subfamily Betaherpesvirinae are particularly suitable for vaccine development as they are highly specific for their natural host species, infect a large proportion of their host population, and cause mild infections in healthy individuals. The Natal multimammate mouse (Mastomys natalensis) is the natural reservoir of Lassa virus, which causes deadly hemorrhagic fever in humans. M. natalensis was recently reported to harbor at least three different cytomegaloviruses (MnatCMV1, MnatCMV2, and MnatCMV3). Herein, we report the molecular cloning of three complete MnatCMV genomes in a yeast and bacterial artificial chromosome (YAC-BAC) hybrid vector. Purified viral genomes were cloned in yeast by single-step transformation-associated recombination (STAR cloning) and subsequently transferred to Escherichia coli for further genetic manipulation. The integrity of the complete cloned viral genomes was verified by sequencing, and the replication fitness of viruses reconstituted from these clones was analyzed by replication kinetics in M. natalensis fibroblasts and kidney epithelial cells. We also found that neither parental nor cloned MnatCMVs replicated in mouse and rat fibroblasts, nor did they show sustained replication in baby hamster kidney cells, consistent with the expected narrow host range for these viruses. We further demonstrated that an exogenous sequence can be inserted by BAC-based mutagenesis between open reading frames M25 and m25.1 of MnatCMV2 without affecting replication fitness in vitro, identifying this site as potentially suitable for the insertion of vaccine target antigen genes.IMPORTANCECytomegaloviruses (CMVs) recently discovered in the Natal multimammate mouse (Mastomys natalensis) are widespread within the M. natalensis population. Since these rodents also serve as natural hosts of the human pathogen Lassa virus (LASV), we investigated the potential suitability of M. natalensis CMVs (MnatCMVs) as vaccine vectors. We describe the cloning of three different MnatCMV genomes as bacterial artificial chromosomes (BACs). The replicative capacity and species specificity of these BAC-derived MnatCMVs were analyzed in multiple cell types. We also identified a transgene insertion site within one of the MnatCMV genomes suitable for the incorporation of vaccine target antigens. Together, this study provides a foundation for the development of MnatCMVs as transmissible MnatCMV-based LASV vaccines to reduce LASV prevalence in hard-to-reach M. natalensis populations and, thereby, zoonotic transmission to humans.

Keywords: DNA virus; Lassa virus; Mastomys natalensis; bacterial artificial chromosome; cytomegalovirus; herpesvirus; muromegalovirus; recombination; yeast artificial chromosome.

Fig 1

STAR cloning of MnatCMV2. (A) Linearized cloning vector and vDNA from MnatCMV2 virions were used to transform yeast spheroplasts. Homology hooks (60 bp, blue and green) for recombination were homologous to regions 110 bp upstream and downstream from the vDNA termini. Circular DNA consisting of the vector and the long unique region from vDNA was isolated from yeast and transferred to E. coli. (B) BAC DNA originating from two independent yeast clones isolated from E. coli clones was digested with NotI or HindIII and analyzed by gel electrophoresis with vDNA as a reference. M, DNA size markers. Vector-vDNA junction fragments (arrowheads) and genome terminal fragments (asterisks) absent from the circular BAC clones are indicated.

Fig 2

Repair of MnatCMV2 BAC via en passant mutagenesis. (A) The incomplete MnatCMV2 BAC (clone 2B), which contained the long unique region (LUR) without the TRs, was repaired by two rounds of en passant mutagenesis. (I) Repair of the left terminus of the MnatCMV2 genome using a linear DNA fragment containing the missing 80 bp of the LUR region (green), two TRs (black), and an I-Sce/Kan cassette (yellow) flanked by an 80 bp duplication (a and a’). Sequences homologous to H1 (dark green) and the pCC1BAC-his3 vector (orange) were used for recombination. The Kan cassette was subsequently removed in the second step of en passant mutagenesis. (II) Repair of the right terminus of the MnatCMV2 genome was performed in the same way as above. The final construct contains the full-length MnatCMV2 genome with a copy of TRs at each end. (III) Complete linear MnatCMV2 genome after cleavage by the viral terminase. (B) Restriction fragment length analysis of BAC DNA before and after repair of the MasCMV2 genome termini. The unrepaired MnatCMV2 BAC (clone 2B) lacking the genomic termini and two repaired BAC clones (2B-1 and 2B-2) obtained after two rounds of BAC mutagenesis are shown. Arrowheads indicate genome differences resulting from the insertion of left and right genome termini. M, DNA size marker.

Fig 3

STAR cloning of MnatCMV1 and MnatCMV3. (A) Linearized pCC1BAC-his3 cloning vectors with homology hooks for vDNA isolated from MnatCMV1 and MnatCMV3 virions were used for the transformation of yeast spheroplasts. The homology hooks consisted of the first and last 60 bp of the viral genome: 30 bp of the long unique region (green or blue) and a 30 bp TR. (B) Restriction fragment length analysis of MnatCMV1 and MnatCMV3 BACs. vDNA is included for comparison. Vector-vDNA junction fragments (arrowheads) and terminal genomic fragments absent in the circular BAC clones (asterisks) are indicated. M, DNA size marker.

Fig 4

Multistep replication kinetics of MnatCMVs. (A) MasEFs were infected at a multiplicity of infection (MOI) of 0.1 with the parental MnatCMVs or the BAC-derived rMnatCMVs. (B) MasKECs were infected (MOI 0.5) with the parental MnatCMVs or the BAC-derived rMnatCMVs. Viral titers in the supernatants were determined. Mean ± SEM of three biological replicates are shown. DL, detection limit.

Fig 5

Multistep replication kinetics of MnatCMVs and MCMV for host range analysis. (A) Mouse embryonic fibroblasts (NIH-3T3), (B) REFs, and (C) BHK-21 fibroblasts were infected with MnatCMV1, MnatCMV2, and MnatCMV3 at an MOI of 0.5. (D) MasEFs and (E) MasKECs were infected at an MOI of 0.01 with MCMV-GFP. Viral titers in the supernatants were determined. Mean ± SEM of three biological replicates are shown. DL, detection limit.

Fig 6

Insertional mutagenesis of the MnatCMV2 BAC. (A) The IGR between ORFs M25 and m25.1 was modified by en passant mutagenesis of the MatCMV2 BAC. The M25 and m25.1 ORFs were tagged with HA and FLAG tag sequences, respectively. In a first step, a Kan cassette for selection was inserted in the middle of the IGR. In the second step, the Kan marker was removed. The vertical lines within the IGR indicate the polyadenylation signals. (B) Restriction fragment length analysis of the mutant MnatCMV2 BACs digested with BamHI. The full-length WT MnatCMV2 BAC and the tagged mutants with or without the Kan cassette (2 clones) are shown. M, DNA size marker. Arrowheads indicate the IGR-containing band. (C) MasEFs were infected with the tagged viruses (MOI 0.1), and expression of the M25 and m25.1 proteins was determined by western blot analysis using anti-HA and anti-FLAG antibodies, respectively. Transfected 293T cells expressing m25.1-FLAG served as positive control (T), mock-infected (M), and parental MnatCMV2-infected cells (WT) were used as negative controls. (D) MasEFs were infected (MOI 0.5) with the BAC-derived rMnatCMV2 viruses. Total RNA was extracted at the indicated times post-infection and analyzed by quantitative reverse transcription PCR (qRT-PCR). Mean ± SEM of three independent experiments is shown. (E) MasEFs were infected (MOI 0.5) with the BAC-derived rMnatCMV2 viruses. Viral titers in the supernatants (mean ± SEM of three biological replicates) were determined. DL, detection limit.