Utilization of homotypic and heterotypic proteins of vesicular stomatitis virus by defective interfering particle genomes for RNA replication and virion assembly: implications for the mechanism of homologous viral interference

Abstract

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV(Ind)) are capable of interfering with the replication of both homotypic VSV(Ind) and heterotypic New Jersey serotype (VSV(NJ)) standard virus. In contrast, DI particles from VSV(NJ) do not interfere with the replication of VSV(Ind) standard virus but do interfere with VSV(NJ) replication. The differences in the interfering activities of VSV(Ind) DI particles and VSV(NJ) DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV(Ind) DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV(NJ) DI particles could assemble only with homotypic VSV(NJ) viral proteins, although the genomic RNAs of VSV(NJ) DI particles could be replicated by using heterotypic VSV(Ind) N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.

FIG. 1.

Schematic representation of chimeric pDIs containing bacteriophage λ sequences. The internal region of the 2,322-bp HindIII fragment of λ DNA is shown as a shaded bar. Numbers in the names of the plasmids indicate the length (in nucleotides) of wild-type sequences at the 3′ and 5′ genomic termini of chimeric DI RNAs. The sizes of chimeric DI RNAs encoded from the plasmids are shown at the right side of the plasmids. Eighty-eight-nucleotide deletions (88 nts) in the λ DNA of pNJ_DI(50-50) are shown under the shaded bar. T7P, HDV, and T7T indicate the T7 transcriptional promoter, hepatitis delta virus ribozyme sequences, and T7 transcriptional terminator, respectively. Numbers in the bracket indicate conserved 3′ and 5′ VSV-specific nucleotide sequences of DI particles.

FIG. 2.

Replication of I_DI(50-50) and NJ_DI(50-50) RNAs with combinations of N, P, and L proteins from VSV_Ind and VSV_NJ. The encapsidated positive-sense and negative-sense chimeric DI RNAs were detected by Northern blot analysis. (A) Schematic representation of DI RNA synthesis after transfection. λ-E/E1 and λ-E/E2 represent the riboprobes for the detection of RNA synthesis by Northern blot analysis. (B) Replication of I_DI(50-50) RNA with combinations of N, P, and L proteins from VSV_Ind and VSV_NJ. (C) Replication of NJ_DI(50-50) RNA with combinations of N, P, and L proteins from VSV_Ind and VSV_NJ. Combinations of N, P, and L proteins from VSV_Ind and VSV_NJ are shown in the boxes. The open arrows indicate the RNA synthesized by T7 RNA polymerase and encapsidated by N proteins. The RNA-N protein complexes were immunoprecipitated with anti-VSV sera. I and NJ denote proteins from VSV_Ind and VSV_NJ, respectively.

FIG. 3.

Replication of chimeric DI RNAs in the presence of homotypic and heterotypic N, P, and L proteins. Open block arrows indicate the chimeric DI RNAs synthesized by T7 RNA polymerase and encapsidated with N proteins. VSV polymerase-specific (second strand) RNA synthesis of chimeric DI RNAs in the presence of homotypic and heterotypic N, P, and L proteins is shown in the left panels. Average values relative to I_DI(255-268) and NJ_DI(227-188) from three experiments are shown in the graphs. Error bars represent standard deviations of the mean. (A) Replication of chimeric DI RNAs in the cells expressing VSV_Ind N, P, and L proteins. (B) Replication of chimeric DI RNAs in the cells expressing VSV_NJ N, P, and L proteins.

FIG. 4.

Assembly and maturation of chimeric DI particles using homotypic and heterotypic VSV proteins. The recovered chimeric I_DI and NJ_DI particles from the transfections were concentrated by ultracentrifugation and were amplified by coinfecting BHK-21 cells with either VSV_Ind or VSV_NJ standard viruses at an MOI of 3. VSV_Ind was used as a helper virus for the DI particles recovered by using VSV_Ind N, P, M, G, and L proteins. VSV_NJ was used as a helper virus for the DI particles recovered by using VSV_NJ N, P, M, G, and L proteins. The presence of chimeric DI particles in the culture fluid was determined by infecting fresh BHK-21 cells with the culture fluid from the first amplification and Northern blot analysis using the λ sequence-specific riboprobe λ-E/E2.

FIG. 5.

Interfering activity of chimeric DI particles. The yields of the standard viruses represent the mean value of duplicate plaque assays. (A) Yield reduction of VSV standard viruses by I_DI(50-50). (B) Yield reduction of VSV standard viruses by NJ_DI(227-188). Error bars represent the standard deviation of the mean of three separate assays.

FIG. 6.

Analysis of VSV RNA synthesized in cells coinfected with chimeric DI particles and the standard viruses. (A) Synthesis of I_DI(50-50) genomic RNA and genomic RNA and mRNA of VSV_Ind and VSV_NJ standard virus in the presence of various concentrations of input I_DI(50-50). (B) Synthesis of genomic RNA of NJ_DI(227-188) and genomic RNA and mRNA of VSV_Ind and VSV_NJ with various concentrations of NJ_DI(227-188). Arrows indicate the genomic RNAs of the chimeric DIs. Std, standard virus; L, G, N, and M/P, mRNA of each gene.