Institute for Biochemistry and Protein Research1 and Laboratory of Gene Technology2, Agricultural Biotechnology Center, PO Box 411, H-2101 Gödöllö, Hungary
Center for Molecular Biology and Gene Therapy, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA3
Author for correspondence: István Fodor at Center for Molecular Biology and Gene Therapy.Fax +1 909 478 4177. e-mail ifodor{at}som.llu.edu
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Previously, we constructed a recombinant PrV (designated as vE16lac) containing a single deletion similar to that of LLT2 with the exception that both IE175 genes remained intact (Boldogköi et al., 1998b
). This mutant retained the ability to infect both pigs and mice and exhibited no significant decrease in virulence. Based on these results, we concluded that deletion of one copy of the analysed DNA segment did not alter the neuroinvasive capability of the virus after i.n. or intraperitoneal (i.p.) inoculation of pigs or mice, respectively. In this study, using a mouse model, we examined the virulence of four PrV strains possessing various modifications at both copies of PLAT2 located at the IR of the virus.
Strain Ka (Kaplan & Watter, 1959 ) of PrV was used as a parental virus for the construction of mutant viruses. Propagation of the virus in porcine kidney (PK-15) cells and preparation of viral DNA for the transfection and construction of recombinant viruses were carried out as previously described (Boldogköi et al., 1998a
). The transfer plasmids used for the construction of recombinant viruses contained a lacZ gene expression cassette, pCMVRI-lac, based on the pCMV
vector (Clontech) and the flanking sequences derived from the target region of the PrV genome. The PrV BamHI-8' fragment extending from -3218 to +1680 (Fig. 1a
) and containing PLAT2 was subcloned into pRL425 and pRL525 (Elhai & Wolk, 1988
), resulting in pB8'-425 and pB8'-525, respectively. Use of these two vectors containing different polylinker regions facilitated our subsequent cloning procedures. Transfer plasmid pIRlac was constructed by converting the DraI recognition site at the putative TATAA box of PLAT2 to EcoRI via linker insertion in pB8'-525, followed by ligation with the EcoRI fragment of pCMVRI. Transfer plasmid pdIRlac was constructed from B8'-425 by deleting the 757 bp DraISmaI DNA fragment in multiple steps and replacing it with an EcoRI site. Recombinant virusesvIR-lac and vdIR-lac were generated by co-transfection of viral DNA to PK-15 cells with pIRlac or pIRdlac, respectively. As a result of homologous recombination and the equalization process (Rall et al., 1992
), the constructed recombinant viruses contained two copies of the lacZ cassette. Viruses expressing
-galactosidase (lacZ) were screened on the basis of their blue plaque appearance in the presence of X-Gal. For the construction of vIR-RI and vdIR-RI, the DNAs of vIR-lac and vdIR-lac were first cleaved by EcoRI restriction endonuclease, resulting in the release of the lacZ cassette, and the fragmented viral DNAs were used for cell transfection. The intact viral genome was reconstituted from the viral DNA fragments by cellular DNA ligases. Viruses containing an EcoRI linker, but missing the lacZ gene, appeared as white plaques. Mutations of both vIR-RI and vdIR-RI were rescued by co-transfection of cloned BamHI-8' fragment with the viral DNAs treated with EcoRI and calf intestinal phosphatase as described (Boldogköi et al., 1998a
). In this system, we exploited the stimulatory effect of double-strand breaks on recombination (Ryan & Shankly, 1996
). The structure of constructed viruses shown in Fig. 1(a)
was confirmed by Southern blotting experiments (Fig. 1b
). To determine the possible impact of mutations on virus growth in vitro, PK-15 cells were infected with viruses at an m.o.i. of 0·1 or 10 p.f.u., and the kinetics of virus growth were investigated. The curves shown in Fig. 2
indicate that the growth rates of both mutant and wild-type viruses are similar (Fig. 2
).
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We also analysed the fate of the viruses after sublethal i.p. inoculation into mice (Table 1c). Wild-type virus can be detected in the brain of the mice without causing lethal infection after inoculation with a low dose (102 p.f.u.), while vdIR-RI spreads to the brain after inoculation with a relatively higher dose (103105 p.f.u.) of virus. Although at day 12 the viruses can still be detected in different organs, most of the animals survived the virus infection, indicating that the virus is not able to invade the body and induce lethal infection.
Taken together, we have found a significant difference between mutant and wild-type viruses in virulence and virus spread in i.p.-infected mice and a moderate difference in i.c. inoculation experiments, while the growth rates of both viruses in vitro were similar. Thus, the sequences at the outer region of IRs, including a putative latency promoter, contain a virulence determinant. Deletion of these sequences affects the capability of the virus to spread from the peripheral neurons to the CNS and, to a smaller extent, from the CNS to the peripheral organs. However, our results do not prove that PLAT2 specifically controls the neuroinvasive capability of the virus because the reduced neurotropism may be the result of impaired virulence of the virus as well.
On the basis of our previously published data and results presented here we can conclude that mutations in the PLAT2 region affect virulence only if they occur in both copies of the IRs. This result is in disagreement with those reported by Dean et al. (1995, 1996 ). There are several possible explanations for the contradictory results obtained in these two laboratories: (1) theoretically, LLT
2 virions could have two alternative forms of genetic structure depending on the outcome of the deletion involving the 3'-end of both IE175 genes. If the mutation caused a lethal effect, in the mature viruses only the Tr segment would be intact, while the majority of viral concatemeric DNAs would contain mutations in both copies of the IE175 genes (in most cases a process termed equalization renders the IR structures identical). However, due to imperfection of equalization in each cycle of replication a small percentage of the Trs would retain their intact genotype. Since the IE175 gene is essential for the virus life-cycle, only virions containing an intact Tr could be infectious. Although, according to our unpublished results, the IE175 activity was found to be normal, a certain portion of IE175 mRNAs must have been mutated and thus, encoded non-functional protein molecules. Lower amounts of functionally active IE175 protein produced by LLT
2 could account for the reduced virulence of this virus. (2) Alternatively, if the deletion was non-lethal for the virus, all mature virions would be mutated in both copies of the IE175 gene. Therefore, the inability of LLT
2 to invade the CNS from the periphery could be explained by the reduced virulence caused by the mutation. Both scenarios presented above are based on results published by Rall et al. (1992)
. (3) The DNA segment located between the stop codon of the IE175 gene and the SmaI restriction site at position 1125 is intact in vE16lac, but it is deleted in LLT
2. (4) For the construction of mutant viruses Chang and coworkers used parental strain Indiana-Funkhauser, while we used strain Kaplan.
A tenfold increase in the LD50 of vIR-RI after insertional inactivation of the putative TATA box of PLAT2 indicates that it may function as a promoter acting at productive infection. Most likely, it controls the expression of an antisense RNA which down-regulates IE gene activity. In this study we have not examined whether PLAT2 plays a role in the latent phase of virus infection. It is pertinent to ask whether the insertion of the intact gene into another genomic location restores the virulence of the mutant virus. We have not investigated this possibility for the following reasons: (1) it is unlikely that the DNA region located downstream of PLAT2 contains the information for a protein. Such a protein encoded on the complementary DNA strand that includes a highly extended (more than 5 kb!) antisense open reading frame (A-ORF; Cheung, 1991 ) would be unique in genetics. The possible origin of non-functional long overlapping ORFs in GC-rich organisms, like PrV, was discussed in our previous papers (Boldogköi & Murvai, 1994
; Boldogköi et al., 1994, 1995
). (2) The A-ORF sequence is co-localized with the IE175 gene on the antisense and the sense DNA strands. Its insertion into another genomic location and thus, formation of three copies of the DNA segment, would cause genomic instability mediated by homologous recombination. In another possible experimental design, both copies of the whole A-ORF could be deleted, and then, a single copy under control of an intact PLAT2 would be reinserted into a different genomic locus. However, in this case we would face serious problems in the interpretation of the results. Indeed, the construct could not be considered as a control for our study because in our work the PLAT2, and not the entire A-ORF, was mutated. In addition, the LLT, extending from the 3'-end of the EPO gene to the 5'-end of the IE175 gene, would also be disrupted by this procedure.
Furthermore, in our previous study we reported that vE16lac could revert with a low frequency to wild-type Irs (Boldogköi et al., 1998b ), but Dean and co-workers (Dean & Cheung, 1995
; Dean et al., 1996
) did not detect LLT
2 revertants, although both viruses had similar genomic struc-tures. We also showed that a low rate of restoration of the Ir segment had little effect on the virulent phenotype of vE16lac.
Finally, we expected that insertion of a lacZ cassette into the vIR-lac would cause drastic structural changes to the promoter and completely abolish its activity. Instead, we found that the difference in LD50 values of vIR-lac and vIR-RI is not significant, but the virulence of vIR-lac is significantly greater compared with vdIR-RI and vdIR-lac. These data indicate that besides its promoter function PLAT2 may have other functions involved in the life-cycle of the virus. Further studies are needed to elucidate the precise function of this region.
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References |
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Received 3 June 1999;
accepted 14 October 1999.