Defective Interfering Semliki Forest Virus Research Articles

Deletion analysis of a defective interfering Semliki Forest virus RNA genome defines a region in the nsP2 sequence that is required for efficient packaging of the genome into virus particles.

The 1,244-nucleotide genome of Semliki Forest virus (SFV) defective interfering (DI) RNA 19 (DI-19) is coterminal with the infectious genome and contains two major deletions. One deletion removes the end of the nsP1 gene and the beginning of the nsP2 gene, and the other removes the end of the nsP2 gene, the nsP3 and nsP4 genes, and all of the structural protein genes (M. Thomson and N. J. Dimmock, Virology 199:354-365, 1994). Like all DI SFV RNAs, DI-19 contains three regions that are conserved. Region a comprises the 5' terminus continuous with part of the nsP1 gene, region b comprises a central part of the nsP2 gene, and region c comprises the 3' terminus and the associated untranslated region. A deletion analysis of the 265-nucleotide b region (nucleotides 679 to 943, inclusive) was undertaken to determine its role in genome replication and packaging into DI virus particles. Deleted plasmids were constructed and transcribed, and the resulting DI RNAs were transfected into SFV-infected BHK cells. Putative progeny DI virus particles that had been released into the tissue culture fluid were then serially passaged in new monolayers together with added high-multiplicity SFV, and cells and tissue culture fluids were tested for the presence of DI RNA by reverse transcription-PCR. DI RNA that had all of the b region deleted was replicated well in BHK-21 cells, as shown by the presence of large amounts of negative-sense DI RNA and an increase in the amount of positive-sense RNA in the cytoplasm, but was packaged very inefficiently, as indicated by very low amounts of DI RNA in the tissue culture fluid. The genome of a deletion mutant that retained the 3' 224 nucleotides of region b was packaged successfully, but one that retained only the 5' 41 nucleotides was not detected in the tissue culture fluid. These and other data suggest that nucleotides 720 to 777 of region b are of particular importance in the packaging process. This finding agrees with data obtained with Ross River virus and contrasts with the well-studied Sindbis alphavirus major packaging signal that is located within the nsP1 gene.

The Genomic Sequence of Defective Interfering Semliki Forest Virus (SFV) Determines Its Ability to Be Replicated in Mouse Brain and to Protect against a Lethal SFV Infectionin Vivo

We have recently cloned and sequenced two genomes of defective interfering (DI) Semliki Forest virus (SFV), DI-6 (2146 nt), and DI-19 (1244 nt). These are similar in that both contain two large central deletions (encompassing the 5′ part of the nsP1 gene and the 3′ part of the nsP2 gene and all of the structural genes), and all the sequence of the latter is represented in the genome of SFV DI-6. RNA was transcribed from both and transfected into SFV-infected BHK-21 cells. RT–PCR analysis of tissue culture fluid harvested 18 h after transfection suggested that SFV DI virions had been rescued from the cloned genomes. Unlike the genomes of noncloned DI SFV, these genomes bred true for at least 7 serial passages. Cloned DI-6 and DI-19 viruses interfered to a similar extent with the multiplication of SFV in cultured cells, but only DI-19 protected mice from a lethal intranasal dose of SFV. Further investigation by RT–PCR analysis showed that DI-19 but not DI-6 genomes were replicated in mouse brain after direct intracerebral injection of DI virus together with an excess of infectious helper SFV. Thus the replication and hence antiviral activity of two closely related DI SFV genomes appears to be exquisitely sequence specific and cell specific. These findings mark a significant step on the way to using DI genomes as antivirals and also may explain why so few animal-protecting DI viruses have been identified.

Common Sequence Elements in Structurally Unrelated Genomes of Defective Interfering Semliki Forest Virus

The polymerase chain reaction was used to amplify defective interfering (DI) Semliki Forest virus (SFV) genomes from tissue culture preparations which had in vitro interfering and in vivo mouse-protecting activity. The products were molecularly cloned and sequenced (pSFVDI-6, 2146 nt and pSFVDI-19, 1244 nt). Comparison with the sequence of the SFV genome showed that both were derived from three noncontiguous regions: the 5′ terminus including the start of the nsP1 coding region, part of the nsP2 coding region, and the 3′ untranslated region. Both clones possessed open reading frames, including one with the potential of encoding truncated versions of nsP1. Transcribed RNAs from pSFVDI-6 and pSFVDI-19 were translated in an in vitro system into polypeptides of M r 27,000 and 25,000, respectively. Although pSFVDI-6 and pSFVDI-19 did not possess the rearranged sequences or extensive repeat regions of previously cloned DI SFV genomes (Lehtovaara et al., Proc. Natl. Acad. Sci. USA 78, 5353-5357, 1981; Lehtovaara et al., J. Mol. Biol. 156, 731-748, 1982), they were derived from similar regions of the SFV genome, suggesting a common sequence requirement for DI SFV particles.

Differential effects of defective interfering Semliki Forest virus on cellular and virus polypeptide synthesis

Defective interfering Semliki Forest virus (DI SFV) inhibited virus RNA and virus polypeptide synthesis in cells coinfected with standard virus but did not delay or alter kinetics of RNA synthesis. Inhibition of polypeptide synthesis was 20-fold greater than that of RNA synthesis which presumably reflected the amplification resulting from cumulative translation of mRNAs. At high concentration, DI virus p12e inhibited the shutoff of host protein synthesis and allowed no synthesis of structural or nonstructural polypeptides. Dilution of DI virus restored the inhibition of host protein synthesis but further dilution was necessary before virus-specified polypeptide synthesis could be demonstrated. Another DI virus (p20a) with the same interference titre as p12e also inhibited shutoff of host protein synthesis but synthesis of virus-induced polypeptides was inhibited differentially: significant amounts of polypeptides comigrating with the structural precursor polypeptide p62 and the nonstructural polypeptide nsp63 were present and the synthesis of nsp90 was little affected at any concentration of DI virus p20a tested. None of the DI viruses tested induced the synthesis of any viral or novel polypeptide. It was concluded that DI SFV preparations have qualitatively different interfering activities in relation to their effects on virus and host cell polypeptide synthesis.

Defective interfering particles of Semliki Forest virus are smaller than particles of standard virus.

By electron microscopy, particles of defective interfering Semliki Forest virus (DI SFV) had a mean diameter of 46.8 nm compared with 55.9 nm for standard virus particles, a decrease of 16%. The difference was confirmed by measurements of the two-dimensional projected areas of DI and standard virus particles. We examined nine different DI virus preparations produced by four to 13 undiluted passages in BHK cells and all were found to contain a majority of the smaller type of particle. Calculation of the absolute number of small particles showed that there were 130 particles per interfering unit measured by the inhibition of virus RNA synthesis. However, a more sensitive assay based on interference with virus protein synthesis gave a particle: interference ratio of 6.5.

Protection of mice infected with a lethal dose of Semliki Forest virus by defective interfering virus: modulation of virus multiplication.

Certain defective interfering (DI) Semliki Forest virus (SFV) preparations completely protected the majority of mice inoculated with a normally lethal dose of SFV, and the surviving mice showed no signs of disease during the period of observation. Depending upon which DI SFV preparation was used, the survivors were resistant to challenge with 100 LD50 SFV (DI SFV p13a), or were completely sensitive (DI SFV p4), the latter having evidently failed to establish a protective immunity. In this report we compared the ability of these two DI SFV preparations to inhibit multiplication of infectious virus in mice inoculated with 10 LD50 SFV. The following conclusions emerged: virus multiplication was profoundly inhibited in the majority of mice treated with either of the DI virus preparations although there was significant multiplication in most tissues, including brain. The number of mice showing evidence of reduced infectivity titres (58%) correlated well with the 60% which survived without disease in lethality experiments. Despite the presence of infectivity, no SFV antigen or histopathological lesions were detected in brain or spinal cord. The DI virus preparations p4 and p13a altered the distribution of infectivity in the mouse in different ways: during the first 2 days of the infection modulated by DI virus p4, the infectivity titres (in brain, olfactory lobes and spleen) were comparatively high, being greater than 1% of those in mice inoculated with standard virus alone. However, from day 3, titres declined precipitously and there was little infectivity in any of the tissues investigated. On the other hand, mice treated with DI SFV p13a had, over the entire duration of infection, greatly reduced though significant infectivity in brain, olfactory lobes and spleen and very little infectivity in serum. In a minority of mice (14.5%), DI virus p13a altered the distribution of infectivity between different tissues so that there was significantly decreased virus in just one or two of the four tissues investigated, suggesting that the infection was being subtly modulated by the DI virus. Interference assays failed to detect DI SFV in any tissue samples although the effects of DI virus on infection in the mouse were obvious.

Defective interfering Semliki Forest virus populations are biologically and physically heterogeneous.

This study demonstrates that populations of defective interfering Semliki Forest virus (DI SEV) are heterogeneous particularly in respect of their interference properties. Interference was quantified by two assays, one measuring inhibition of the yield of infectious progeny virus, and the other measuring reduction in virus-directed RNA synthesis; for 11 different DI SFV preparations a ratio of the two interference titres was calculated. These ratios varied up to 46-fold indicating that each DI virus preparation contained an interference activity that varied independently of the other. However, sister stocks made from the same parental inoculum had similar properties. The effects of different DI virus preparations on other parameters (virus polypeptide synthesis, yield of DI virus and yield of infectious virus) were investigated using inocula with interference titres standardized by either assay. Co-inoculation of L929 cells with 50 p.f.u. SFV showed that these parameters varied independently of each other and of the DI virus inoculum. There was no correlation with the number of undiluted passages each DI stock had received. Direct evidence of physical heterogeneity was demonstrated by metrizamide density gradient centrifugation. Although infecting virus sedimented as a narrow band, DI SFV was distributed over a broad region of the gradient. Its position on the gradient indicated that DI SFV has a higher nucleic acid: protein ratio than standard virus. DI virus progeny obtained by using fractions of the gradient as inoculum were as heterogeneous as the unfractionated parent, confirming that DI viruses retain heterogeneity on passage.

Properties of host and virus which influence defective interfering virus mediated-protection of mice against Semliki Forest virus lethal encephalitis

Variation of the mouse strain, the amount of defective interfering (DI) virus and the amount of infectious Semliki Forest virus (SFV) are all factors which affect the efficiency with which DI SFV prevents the SFV-mediated lethal encephalitis.

The effect of defective-interfering Semliki Forest virus on the histopathology of infection with virulent Semliki Forest virus in mice.

The majority of mice inoculated with a mixture of a lethal dose of virulent Semliki Forest virus (SFV) strain ts+ and defective-interfering (DI) SFV remained completely health, with virus infectivity levels in brain tissue reduced by 99.9%. The results of previous studies had suggested that these effects were primarily the result of the intrinsic interfering capacity of DI virus rather than of host defense responses. Because SFV strain ts+ and an avirulent strain of SFV have clearly distinguishable histopathologic effects in brain tissue, the capability of DI virus to change the virulent into the avirulent form of the disease was examined. Modulation of strain ts+ virus infection by DI virus was accompanied by a complete absence of histopathologic changes despite significant levels of infectious virus and thus differed qualitatively from infection with avirulent SFV. These results provide further evidence that the interference is not mediated through stimulation of an immune cell infiltration.

Prevention of death in Semliki Forest virus-infected mice by administration of defective-interfering Semliki Forest virus.

Adult mice inoculated with Semliki Forest virus (SFV) were protected from a lethal infection of the central nervous system by intranasal administration of defective-interfering (DI) SFV. DI SFV was prepared by eight passages at high m.o.i. in BHK 21 cells. Mice were treated with unpurified, unconcentrated tissue culture fluid which had been u.v.-irradiated to inactivate the infective virus present. Prevention of death was maximal when the DI virus was administered simultaneously with the infecting inoculum, and under the same conditions multiplication of infective virus in the brains of treated mice was reduced by 10(5)-fold. It was shown that DI SFV was propagated in mouse brains followed intranasal inoculation and it was concluded that protection was brought about through the intrinsic interfering capacity of the DI virus.

Sequence relationships between the genome and the intracellular RNA species of standard and defective-interfering semliki forest virus

Oligonucleotide fingerprinting on slabs of polyacrylamide gel (De Wachter & Fiers, 1972) has been used to investigate the sequence relationships between the 42 S RNA genome of standard Semliki Forest virus and the intracellular virus-specified single-stranded RNAs isolated from baby hampster kidney cells infected with standard virus and co-infected with standard virus and defective-interfering particles. For the intracellular RNAs of standard virus-infected cells these studies show that the nucleotide sequence of the 26 S RNA, which acts as messenger RNA for the structural proteins of the virus particle Clegg and Kennedy, 1975a , Clegg and Kennedy, 1975b , is located at the 3′ end of the 42 S RNA and that the 38 S and 33 S RNAs which appear as minor species on RNA polyacrylamide gels run under non-denaturing conditions, are conformational variants of the 42 S and 26 S RNAs, respectively. Studies on the two RNAs of defective-interfering particles isolated from cells co-infected with standard virus and defective-interfering particles indicate extensive sequence homology between the defective-interfering RNAs, and show that both RNAs contain nucleotide sequences present in both the structural and non-structural genes of the 42 S RNA. Furthermore, by determining the order (5′→3′) of the characteristic oligonucleotides of the 42 S RNA, and by digestion with snake venom exonuclease, it is concluded that the nucleotide sequences of the defective-interfering RNAs are derived from the 5′ and 3′ extremities of the 42 S RNA; about 75% from the 5′ end and 25% from the 3′ end. The significance of this observation in relation to the genesis of defective-interfering particles and to the mechanism of interference by the defective-interfering RNAs is discussed.