Institute for Experimental Pathology, University of Iceland, Keldur, IS-112, Reykjavík, Iceland1
Author for correspondence: Valgerdur Andrésdóttir. Fax +354 567 3979. e-mail valand{at}rhi.hi.is
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Abstract |
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MVV was originally isolated from the brains of visna-affected sheep and was grown in sheep choroid plexus (SCP) cells, since visna lesions always occurred in the choroid plexus and virus was readily isolated from this tissue (Sigurdsson et al., 1960 ). However, not all field isolates of MVV grow well in SCP cells (Narayan et al., 1982
; Querat et al., 1984
) and it has been demonstrated that virus isolates from maedi-affected lungs grow more slowly in SCP cells than do the original visna strains (Andrésdóttir et al., 1998
; Thormar, 1965
). In a previous study, the nucleotide sequence of the env gene and the LTR of a field strain, 1071, which was isolated from a maedi-affected lung, was determined (Andrésdóttir et al., 1998
). The sequenced clone contained only one copy of a 43 bp sequence in the LTR that is duplicated in the visna strains that have been sequenced (Andrésson et al., 1993
; Braun et al., 1987
; Sonigo et al., 1985
; Staskus et al., 1991
). However, half of the PCR products from the Hirt supernatant (Hirt, 1967
) of virus-infected cells were larger, which would possibly indicate a duplication in the LTR.
In this study, we cloned the two size variants of LTR from the maedi strain in the infectious molecular clone of visna virus, KV1772kv72/67 (Andrésson et al., 1993 ) and tested the resulting chimeric viruses for replication in various cell types.
The two LTR bands from maedi strain 1071 were isolated from an agarose gel, cloned in M13 and sequenced. The two sizes of LTR differed from each other only in that the larger one had a 53 bp duplication. The sequence that was duplicated overlapped the 43 bp repeat in the visna strains by 34 bp (Fig. 1). The duplication or lack of duplication is unlikely to be an artefact from the PCR, since we have never detected this kind of artefact in more than fifty PCR products from cloned LTRs that we have sequenced.
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On further passage in SCP cells, there was selection for the variant with the duplication, and this was already an indication that the duplicated sequence enhanced growth in SCP cells. In the SCP cells infected with 1071VA4, the variant with the 53 bp duplication, there was no indication of recombination between the two LTRs. We then isolated VA3, without the duplication, by end-point dilution of the supernatant from the transfected FOS cells in blood-derived macrophages. PCR products from both LTRs were sequenced, with primers corresponding to base pairs 84608481 (forward) and 91779156 (reverse) to amplify the 3' LTR and 88658876 (forward) and 522504 (reverse) to amplify the 5' LTR, with bp 88658876 as a sequencing primer. Nucleotide numbers are as published for KV1772kv72/67 (Andrésson et al., 1993 ). The sequence analysis revealed no heterogeneity in the LTRs.
As sheep blood-derived macrophages were permissible for all three viruses, these cells were used for preparation of virus stocks. Macrophage cultures were established as described previously (Torsteinsdóttir et al., 1997 ) and infected with supernatants from the transfected FOS cells. Virus was harvested when CPE was clearly visible and equal amounts of virus (as determined by measuring RT activity, usually 100010000 c.p.m.) were used to infect fibroblasts from sheep longitudinal vertebral tendon, SCP cells and FOS cells as well as macrophage cultures. The m.o.i. was estimated by using a titrated KV1772kv72/67 stock as a standard and determining the correlation between TCID50 and RT activity. Growth was monitored by taking samples twice daily and measuring RT activity. Multiplicities of 0·5 and 5 were used. Fig. 3
shows growth curves when the cultures were infected with an m.o.i. of 0·5.
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We have shown here that cellular tropism of MVV is determined by the LTR. The results do not allow us to conclude at what level of the replication cycle the LTR exerts its effect. However, the region in the LTR that needs to be duplicated for replication in SCP cells, FOS cells and sheep fibroblasts contains a number of known transcription factor-binding sites, including redundant AP-1 sites, a CAAAT sequence and a PEA-2 site, which has been implicated in the replication of EIAV in fibroblasts (Payne et al., 1999 ) and has also been suggested to be important in controlling MVV replication (Sutton et al., 1997
) (Fig. 1
). In the visna strain 1514, an AP-1 site proximal to the TATA box has been shown to be important for transcription in macrophages (Gabuzda et al., 1989
; Hess et al., 1989
). The receptor for MVV has not been identified, but there are indications that the receptor may be a relatively common molecule and that cell tropism is determined by cell-specific replication factors. This notion was supported by the findings of one study, that ovine lentivirus could enter a variety of cell types in vivo, but productive infection was restricted to macrophages (Brodie et al., 1995
).
The importance in vivo of the broadening of cell tropism is unclear. The visna virus strains characterized in our laboratory were originally isolated from the choroid plexus and, although the macrophage is the main target cell of MVV, neurotropic strains may need to grow in other cell types in order to cross the bloodbrain barrier. Although infrequent, productive infection has been demonstrated in endothelial cells (Georgsson et al., 1989 ). In this context, it is interesting to note that it has been shown that neuroinvasiveness of murine retroviruses is determined partly by the LTR (Portis & Lynch, 1998
). The molecularly cloned visna virus used in the present studies, KV1772kv72/67, is highly neurotropic and neurovirulent in vivo (Andrésson et al., 1993
). It is therefore well suited for studying neurotropism and neurovirulence. Studies on the role of the duplications in the LTR in neurotropism and pathogenicity are under way in our laboratory.
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Acknowledgments |
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References |
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Received 18 January 2000;
accepted 3 May 2000.