Journal of Virological Methods, 36 (1991) 129-140 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
129
VIRMET 1277
Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester David R. Sargan,
Neil J. Watt and Ian McConnell
Department of Veterinary Pathology, University of Edinburgh, Edinburgh, U.K. (Accepted
13 September
1991)
Summary The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeticiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15~11of sample, is rapid, and is able to detect virus at titres below 103/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection. Reverse transcriptase;
Lentivirus; Radiolabelling; Cell harvester; Visna/maedi; HIV
Introduction Intensive research on retroviruses of the lentivirus subgroup has been triggered by the discovery of the human immunodeficiency viruses HIV I and II, the causative agents of the disease AIDS, and their identification as Correspondence to: D.R. Sargan, Dept. of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 lQH, U.K.
130
members of this subgroup (reviewed in Narayan and Clements, 1989.) Lentiviruses can be distinguished from most other mammalian retroviruses by the divalent cation and template specificities of their RNA-dependent DNA polymerases (reverse transcriptases) (Lin and Thormar, 1970; Goodman and Spiegelman, 1971; Stone et al., 1971; Hoffman et al., 1985). Assays for these enzymes can be a sensitive and rapid test for the presence of viruses in this group (Spira et al., 1987; Gregersen et al., 1988). We have investigated the characteristics of the reverse transcriptase of the sheep lentivirus visna/maedi virus (de Boer, 1975) in tissue culture media and cell lysates by using a microscale semi-automated assay. The assay measures the inco~oration of radiolabelled thymidine triphosphate into nucleic acids using an oligo(dT) primer and poly(rA) template. Incorporated nucleotides are distinguished from unincorporated using either acid precipitation (if protein concentration in the sample is low) or binding to cation exchange paper (if protein concentration in the sample is higher). The assay is performed in microtitre plates, similar to that reported by Gregersen et al. (1988) but unlike their assay, a cell harvester is used to filter and process assay supernatants, allowing handling of several hundred samples at once. We have used the assay to demonstrate directly the presence of viral reverse transcriptase within infected cells from permissive cultures within 36 h of visna infection in vitro. The assay cannot, however, cope with the very high protein concentrations (> 50 mg/ml) found in lymph and sera from infected animals. Both nonspecific RNase and DNase activities in samples may interfere with the assay, and we have developed protocols to minimise these effects. Materials and Methods Materials
E3H]TTP (20 C/1 mmol) and [32P]TTP (400 Ci/mmol) were from Amersham. Synthetic otigo- and polynucleotides and human placental ribonuclease inhibitor were from Pharmacia. DEAE paper was from Whatman. Glass fibre filters (45 micron pore size) were from Skatron AS. Tissue culture media and plasticware were from Gibco. Sheep DNA was prepared by standard methods from peripheral blood of Scottish Blackface x Finn sheep. All other reagents were of Analar or equivalent quality. Methods Visna virus samples
Visna virus was grown in primary cultures of choroid plexus cells (Weybridge cell line) between passages 15 and 30 in Dulbecco’s Minimal Essential Medium containing 1% foetal calf serum. Cells were infected at MOI below 0.05 with visna virus of strain 1514 (Sonigo et al., 1985) or one of two
131
British strains designated K184 and EVl (Sargan et al., 1991). Tissue culture supernatants were harvested from infected cells for use as sources of virus at various times. Larger cell debris was removed from these supernatants by centrifugation for 10 min at 5000 x g, and the clarified virus containing media either used directly in the assay or subjected to further purification by a modification of the method of Hoffmann et al., 1985. Virus was recovered from 12-ml samples of the clarified media by centrifugation for 40 min at 192 000 x g and resuspended in 250 ~1 TNE (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA). Pooled samples were layered onto a 3-ml 15% sucrose cushion in TNE above a l-ml pad of 65% sucrose in TNE and spun at 100 000 x g for 1 h. A 0.5-ml sample from the interface of the 2 sucrose solutions was then diluted to 2 ml and loaded onto a linear lo-ml 2065% sucrose gradient in TNE. This was subjected to centrifugation for 16 h at 192 000 x g and 20 drop fractions were collected by upward displacement for reverse transcriptase assay. In vitro infected choroid plexus cells for assay of cell-associated RT activity were stripped from culture vessels by scraping and washed twice with Hanks Balanced Salt Solution (HBSS) containing 1% foetal calf serum and twice in HBSS alone. They were resuspended at 5 x lo6 cells/ml in the same buffer. 0.5% Triton X-100 was added and the cells lysed by homogenization. Cells were then diluted to 1 x 106/ml using HBSS prior to assay for reverse transcriptase. Primary cultures of synovial libroblasts from CAEV-infected animals were maintained in culture for 10 days prior to treatment as described above. Reverse transcriptase assay Samples for assay (15 ~1) were placed in wells of 96-well U-bottomed ELISA plates. 10 ul of 2.5 x reaction mix (2.5 x reaction mix = 375 mM KCl, 25 mM MgC12,25 mM Tris-HCl, pH 8,5 mM DTT, 1.25 mM EGTA, 0.25% Triton X100, 62.5 ug/ml BSA, 75 ug/ml poly(rA).oligo(dTiz_is), 5% (v/v) ethane diol (deionized), 250 uCi/ml (or as stated in text) [3H]TTP (concentration 12,.5 uM in all cases). A 5 x reaction mix stock without template or primer and DTT was routinely stored at - 20°C for periods of several weeks. The plate was then incubated for 1 h at 37°C or as stated in the text. The assay was stopped and harvested by one of two methods: (I) TCA precipitation assay 100 ul of 10% (w/v) trichloroacetic acid (TCA) containing 50 ug/ml yeast RNA (Sigma) was added to each well and the plate left on ice for 15 min. Samples were then harvested onto a glass fibre mat filter (Skatron) using a Titertek cell harvester. Wells and filters were washed 3 x with 5% (w/v) TCA, 30 (w/v) sodium pyrophosphate and 2 x with 70% ethanol and dried. Filter discs were cut out and counted in Optisafe (a proprietary scintillant, supplier LKB) in a beta counter. (2) DEAE-paper
assay
100 ul of 10 x TE (100 mM Tris-HCl,
pH 8, 10 mM
132
EDTA) were added to each well and samples were harvested onto Whatman DEAE paper (DE81) supported by a glass-fibre filter on the cell harvester. The wells and filter were sequentially washed three times with 10 x TE containing 0.1% Triton X-100 and twice with 10 x TE containing 0.15 M NaCl and 0.1% Triton X-100. Filters (both DEAE paper and glass-fibre backing) were dried and counted as before. This method proved to have similar sensitivity to method (1) above, but also lower backgrounds in the presence of protein (see Results). Samples for DNA gel electrophoresis were prepared using [32P]TTP in standard assays. After stopping the assay using 100 pl 10 x TE, assays were phenol chloroform extracted and ethanol precipitated, prior to electrophoresis on 8.3 M urea polyacrylamide gels by standard methods. Results Characteristics of the reverse transcriptase of visna virus; salt optima and detergent characteristics
Using the microscale assay and the cell harvester we measured incorporation of [3H]TTP into acid insoluble products by purified visna virus samples under a number of different conditions. Parameters investigated included KC1 and Mg2+ concentrations, template concentration and the ethane diol, EGTA and Triton X-100 dependence of the assay. In all of these assays a 60-min incubation at 37°C of 15 ~1 of a 20 or loo-fold dilution of sucrose gradient purified visna type 1514 from infected WSCP cells was used in a 25 l.d assay. No RNase inhibitor or carrier DNA was used. The results of these assays are shown in Fig. 1. As can be seen the assay has a broad optimum for Mg2+ concentration around 7-10 mM, and an optimum for KC1 of 150 mM. Optimum Mn2+ concentration in the assay is 0.6 mM to 0.8 mM, but this divalent cation gives much lower maximum incorporation than Mg2+ (Table TABLE 1
Template for assay
cpm in assay
None poly (rA).oligo poly (dA).oligo oligo (dTrz-1s) poly (rA).oligo poly (rA).oligo
1366+46 19678+770 1519&493 677 f 89 35844+6009 4 643 + 334
(dTrs_rs) (dTrs_rs) (dTr&, + 0.5 mM EGTA (dTrz_rs), + 0.8 mM Mn’+, no Mg2+
Sucrose gradient purified visna virus strain K184 was diluted 20 times in TNE, and duplicate samples were assayed using the TCA precipitation assay, in the presence of 75 ug/ml of each template as shown. Except where stated these assays were in 5 mM MgC12 without EGTA. Mean counts incorporated in a 60-min assay are shown.
0' 0
loo
200
300
400 KCI mM
0
2-5
5
7-5 MgCIz
10
mM
Fig. 1. Characteristics of the reverse transcriptase assay. Assays were performed on various samples of sucrose gradient puriiied visna virus type 1514, diluted 20 or 100 times with HBSS, using 2.5 @ci 13H]‘ITP per assay and reaction conditions as stated. (A) Effect of varying KC1 concentration; (B) effect of varying MgC& concentration; (C) effect of varying pH.
1). Maximum in~o~oration occurred at pH 8.25. In~o~oration was totally dependent on the presence of Triton X-100, but independent of its concentration in the range 0.05-0.5%. The assay was activated by EGTA (see below). Ethane dial had a slight activating effect. Incorporation in the assay was maximal at template concentrations above 10 ug/ml in these conditions, These results show that visna virus reverse transcriptase has very similar reaction characteristics to those already published for HIV (Hoffman et al., 1985; Rey et al., 1984; Cheng et al., 1987) The assay measures a reverse ~r~nscriF~ase activity
To control for the possibility that the assay was measuring DNA polymerase activity, a poly(dA) template was substituted for the poly(rA) template, again
134
using an oligo(dT) primer (Table 1). Little activity was seen using this template. Similarly little activity was seen in the absence of template but in the presence of primer, suggesting that no terminal transferase activity was being detected by the assay. Similar results were found when clarified tissue culture supernatant from visna infected cells or lysed infected cells were used.
Sensitivity
and time course of the assay
TABLE 2
Gross cpm in assay Sample dilution
Cell harvester
Filtration
l/l l/3 l/9 l/21 Mock-infected,
6 218 *438 4 340 + 251 1548+116 855& 146 110+26
6613k530 4 409 + 290 3 631+253 2154k156 1218
l/l
manifold
Tissue culture supernatants from visna virus type EVl infected WSCP cells (titre = 3.8 x 10’ TCIDSo/ml-‘) were diluted as shown with HBSS and assayed (0.5 pCi [3H]TTP, 60 min). Assays were filtered using either the Flow cell harvester or a Millipore filtration manifold, and filters washed extensively using in both cases 5% TCA, 3% sodium pyrophosphate followed by 70% ethanol.
1
2
3
4
18h
. Time
Fig. 2. Time course of incorporation in the assay. Visna type 1514 from various sources was incubated in triplicate assays with 13H]TTP and the assay stopped at various times. Incorporation into acid-soluble material was measured. (0) Clarified tissue culture medium, no RNase inhibitor; (a) clarified tissue culture medium, 0.25 U/assay RNase inhibitor; (A) sucrose gradient purified virus diluted 1:lOO with HBSS.
NO _
Ca2+
II-L-!3 to 30 100
Fig. 3. (A) Sensitivity of the reverse transcriptase assay. Titres of different strains of virus in tissue culture media were measured by serial dilution and plating onto choroid plexus cells. The same samples were used in reverse transcriptase assays, using 2.5 $i [3H]‘ITP and reaction conditions as stated. Harvesting was by acid precipitation. Points show mean inco~ration in triplicate assays. Error bars are omitted for clarity. (A) Visna strain K184; (Cl) a British CAEV isolate; (e) visna 221050; (0) visna strain 1514; (II) visna strain EVl. (B) Sizes of reverse transcriptase products in the assay. Sucrose gradient purified visna virus strain 1514 was diluted by the amounts shown with HBSS and used in 2-h assays with [s2P]TTP (2.5 @i/assay, 100 Ci/mmol, 12.5 ul assays). Products of the assay were extracted with phenoi/chlorofo~ denatured and sized on 7.5% ~lyac~l~ide 8.3 M urea gels. Lanes l-4: assay in the presence of 0.5 mM EGTA, no Car-+; lanes 5-8: as l-4, but 2.0 mM Ca2+. Sizes of marker DNA fragments are shown.
To validate the use of the cell harvester to filter assays, duplicate assays were performed on visna-infected tissue culture supernatants which were harvested using either the cell harvester or a Millipore barrel filter apparatus and Whatman GF/C type filters. Incorporation as measured by either assay was very similar (Table 2), but background as measured on the cell harvester was much lower. Sucrose gradient purified visna virus (strain 1514) or clarified tissue culture
136
supernatants containing virus were used in assays to investigate the effect of different incubation periods. When no RNase inhibitor was present incorporation into acid precipitable material plateaued after 60 min in tissue culture supernatants. However, when purified virus samples were used, incorporation remained linear for 4 h and continued to increase overnight (Fig. 2). Addition of RNase inhibitor to the assay increased incorporation of TTP at later times when clarified tissue culture media were used, but had little effect on purified samples. The sensitivity of the assay was measured by titrating virus present in tissue culture media using HBSS. The titre of infectious virus in the samples was measured by examining the dilutions for ability to induce CPE on WSCP in 96 well plates. Such assays were performed for visna strains 1514 and 221050, and the 2 British visna isolates (K184 and EVI), as well as for a British CAEV isolate (Fig. 3A). Reverse transcriptase activity was measured in 60-min assays at 37°C containing 2.5 PCi [3H]TTP, but in the absence of RNase inhibitor. The sensitivity of the assay varied with strain, the highest sensitivity being found with visna K184. For this virus a strong positive signal (20000 cpm above background) was found to correspond to the presence of lo3 virus TCIDSO/ml with a sample size of 15 ~1. (Note, however that the proportion of defective virions in the assay is unknown.) Sensitivity is improved approx. lo-fold by using [32P]TTP (10 ~Ci~s~ple, 50 Ci/mmol) in a 4-h assay in the presence of placental RNase inhibitor (data not shown). It was noted that the assay was not linear with dilution of the virus. A possible explanation for this observation emerged when the size of transcripts produced in the reaction was measured by electrophoresis (Fig. 3B). When higher dilutions of virus samples were used, transcript sizes increased. These effects were particularly marked in virus samples from tissue cultures in which cells had been lysed by the virus (data not shown). Competition for template or for free nucleotide triphosphates is not likely to cause these results because template concentration could be reduced 2.5-fold without affecting the level of incorporation into the assay (data not shown) and total incorporation was less than 4% of supplied nucleotide t~phosphates in any sample. Because the assay is linear with time when purified virus is used, it is unlikely that viral RNase H activity destroying the reaction template is the cause of non-linearity in nonpurified samples, but possible that nonspecific deoxyribonuclease and ribonucleases are present in these samples, which at high concentrations destroyed the reaction products. When EGTA was omitted from the assay the size of synthesized transcripts decreased, and it was possible to increase the linearity of the assay at low dilutions of virus by dialysis of virus samples against TNE. These observations are consistent with the presence of a calciumdependent deoxyribonuclease similar to many of those purified from eukaryotic cells (Duerksen and Connor, 1978). We therefore added 100 pg/ ml of purified DNA from sheep thymus to each reaction as a carrier. In some, though not all samples, this increased the linearity of the reaction with respect to dilution of the virus sample, and the total counts inco~orated in the
137
reaction at high virus concentrations. In contrast, addition of Ca*+ to the assay reduced transcript sizes (Fig. 3B). Measurements
of reverse transcriptase
in infected cells
We have attempted to measure reverse transcriptase activity in visna-infected cell populations and body fluids from visna and CAEV-infected animals. Harvesting of the assay using TCA precipitation and filtration onto glass-fibre filters led to unacceptably high backgrounds with these materials. We therefore developed an alternative means of harvesting the assay. The assay was stopped with 100 ul 10 x TE and harvested onto DEAE paper backed by a glass-fibre filter for strength, using the cell harvester as before. Sequential washes with salt solutions were then used to remove unincorporated nucleotides. After some experimentation it was found that two washes with 10 x TE, 0.1% Triton X100, followed by two washes with 0.15 M NaCl, 10 x TE, produced high signals combined with relatively low background. This assay allowed detection of reverse transcriptase in extensively washed WSCP cells at short times (36 h) after infection in vitro (MO1 = 0.1-1.0) (Table 3). This was considerably before these cells showed any cytopathic effect, though after the first appearance of viral transcripts (Sargan et al., in preparation). The assay also allowed detection of cell-associated reverse transcriptase in primary cultures of synovial libroblasts from CAEV-infected goats. Though the DEAE paperbased assay allowed detection of cell-associated virus, it was still unable to cope TABLE 3 Reverse transcriptase
assays on cell lysates
Sample source
Visna Visna Visna Visna Visna Visna Visna Visna CAEV CAEV
1514 from WSCP, TC fluid (TCA) 1514 from WSCP, TC fluid (DEAE) 1514 in WSCP cell lysates, 8 h p.i. 1514 in WSCP cell lysates, 24 h p.i. 1514 in WSCP cell lysates, 36 h p.i. 1514 in WSCP cell lysates, 48 h p.i. 1514 in WSCP cell lysates, 5 days p.i. 1514 in WSCP cell lysates, 5 days p.i. (TCA) goat synovial membrane (expt. 1) goat synovial membrane (expt. 2)
cpm in assay Virus-positive sample or infected animal
Control sample or uninfected animal
2 202 f 384 3 136k50 202k25 209k9 462 f 58 922 f 53 20 490 + 2 960 21414k3653 1057+66 3 596 + 293
290+61 106+48 211+39 NA NA NA 387 f 209 1127k340 184+35 NA
Rows 1 and 2: a comparison of TCA precipitation and DEAE paper filtration; rows 3-6: detection of cell-associated reverse transcriptase by DEAE paper filtration in WSCP cell populations infected in vitro at various times post infection; rows 7,8 detection of cell-associated reverse transcriptase in lysates of cell populations from CAEV-infected goats (2 independent experiments are shown). Synovial membrane cells from biopsy specimens were maintained in tissue culture for 10 days prior to assay. Cell populations were lysed and prepared for assays as stated. Assays using 1 uCi [ H]TTP were performed and harvested using the DEAE paper method (except rows 1 and 8). Figures represent mean counts incorporated, and standard errors of three samples.
138
with the high levels of protein found in lymph or synovial fluid from infected animals (data not shown). Discussion
A semi-automated and therefore rapid assay for lentivirus reverse transcriptase in fluid media or in lysed cell populations is presented here. The assay is fairly inexpensive to perform, the cost being largely determined by the amount of radioisotope used. Modification of the harvesting procedure in the assay by using DEAE paper and salt washes allows assay of samples with protein concentrations greater than 5 mg/ml, but with slight loss of sensitivity when compared with assay by TCA precipitation. The assay has not been able to detect lentivirus reverse transcriptase in direct measurements of lymph or serum samples, but can detect reverse transcriptase in extensively washed infected cells.
Visna
Present
Ccpel
+
+
+
+
+
+
Fig. 4. Autoradiograph of part of a filter mat from the cell harvester after assay of 6 visna virus positive and 6 negative samples. Tissue culture fluid from infected and uninfected cell cultures was used in 25 ul assays containing 0.5 uCi/assay [‘*P]TTP. After incubation, harvest and wash the filter mat was subject to autoradiography for 2 h at room temperature.
139
The amount of product given by the reaction and the reaction’s linearity with time and virus concentration are increased by the presence of RNase inhibitors and of carrier DNA. This suggests that contamination of viral samples with both DNases and RNases may be a common problem in reverse transcriptase assays. The effect of DNases in particular was increased in virus samples from cell cultures in which marked viral-induced cell lysis had occurred. The effect could also be reduced by titration of the sample into a calcium-free buffer. The assay is best suited to the quantification of virus in large numbers of samples of low protein concentration (for instance tissue culture media derived either from virus culture or from coculture of potentially infected cells with permissive cell lines). Very rapid screening for product formation can make use of [32P]TTP in the assay, and use autoradiographs of the dried filters to detect products of the reaction (Fig. 4). By this method single positives can be found in thousands of samples, and a single worker can easily handle several hundred samples in a day. Although previous reports have shown that the visna reverse transcriptase is Mg2+-dependent, no detailed characterization of the requirements of the enzyme has been presented (Lin and Thormar, 1970; Stone et al., 1971). The reaction optima for sheep lentiviruses and for HIV- 1, bovine immunodeticiency virus (BIV), and caprine arthritis encephalitis virus (CAEV) (in terms of salt concentration etc.) are rather similar (Kashanchi et al., 1991; Hoffman et al., 1985; Clements et al., 1980). (The enzymes have optima for KC1 of around 150 mM, for pH 8.0-8.25, and a preference for Mg2+ rather than Mn2+) An ancestral relationship between HIV-l and the lentivirus group in terms of genome structure and sequence has been previously demonstrated (Gonda et al., 1985; Chui et al., 1985; Sonigo et al., 1985; Querat et al., 1990). The assay may therefore have general application as a method for assessing the presence of lentiviruses in samples when the type of virus has not been fully characterized, or no antibody based test is available.
Acknowledgements
We wish to acknowledge excellent technical assistance from Mr I. Bennet, gifts of viral samples and other materials from Dr M. Dawson and Dr G. Harkiss and to many colleagues for helpful discussions. The research was supported by a project development award from the Wellcome Trust.
References Cheng, Y.-C., Dutschman, G.E., Bastow, K.F., Sargadharan, M.G. and Ting, R.Y.C. (1987) Human immunodeficiency virus reverse transcriptase: general properties and its interaction with nucleoside triphosphate analogs. J. Biol. Chem. 262, 2187-2189. Chui, I.-M., Yaniv, A., Dahlberg, J.E., Gazit, A., Skuntz, SF., Tronick, S.R. and Aaronson, S.A. (1985) Nucleotide sequence evidence for relationship of AIDS retrovirus to lentivirus. Nature 3 17, 366-368.
140 Clements, J.E., Narayan, 0. and Cork, L.C. (1980) Biochemical characterisation of the virus causing leukoencephalitis and arthritis in goats. J. Gen. Viral. 50, 423427. deBoer, G.F., Teopstra, C. and Howens, D.J. (1975) Studies in the epidemiology of maediivisna in sheep. Res. Vet. Sci. 26, 202-208. Duerksen, J.D. and Connor, K.W. (1978) Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases. Mol. Cell. Biochem. 19, 93-l 12. Gonda, M.A., Wong-Staal, F., Gallo, R.C., Clements, J.E., Narayan, 0. and Gilden, R.V. (1985) Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus. Science 227, 173-177. Goodman, NC. and Spiegelman, S. (1971) Distinguishing reverse transcriptase of an RNA tumor virus from other known DNA polymerases. Proc. Nat]. Acad. Sci. USA 68, 2203-2206. Gregersen, J.P., Wege, H., Preiss, L. and Jentsch, K.D. (1988) Detection of human immunodeficiency virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay. J. Virol. Methods 19, 161-168. Hall, J.G. and Morris, B. (1962) The output of cells in lymph from the popliteal node of sheep. Quart. J. Exp. Physiol. 47, 36&369. Hoffmann, A.D., Banapour, B. and Levy, J.A. (1985) Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147, 326335. Kashanchi, F., Lui, Z.Q., Atkinson, B. and Wood, C. (1991) Comparative evaluation of bovine immunodeticiency-like virus infection by reverse transcriptase and polymerase chain reaction. J. Viral. Methods 31, 197-210. Lin, F.H. and Thormar, H. (1970) Ribonucleic acid dependent deoxyribonucleic acid polymerase in visna virus. J. Virol. 6, 702-704. Narayan, 0. and Clements, J.E. (1989) Biology and Pathogenesis of Lentiviruses. J. Gen. Virol. 70, 1617-1639. Querat, G., Audoly, G., Sonigo, P. and Vigne, R. (1990) Nucleotide sequence analysis of SAOMVV, a visna -related ovine lentivirus: phylogenetic history of lentiviruses. Virology 175,434 447. Rey, M.A., Spire, B., Dormont, D., Barre Sinoussi, F., Montagnier, L. and Clermann, J.C. (1984) Characterization of the RNA-dependent DNA polymerases of a new human T lymphotropic retrovirus (lymphadenopathy associated virus). Biochem. Biophys. Res. Comm. 121, 126133. Sargan, D.R., Bennet, I.D., Cousens, C., Roy, D.J., Blacklaws, B.A., Dalziel, R.G., Watt, N.J. and McConnell, I. (1991) Nucleotide sequence of EVI, a British isolate of maedi-visna virus. J. Gen. Virol. 72, 1893-1903. Sonigo, P., Alizon, M., Staskus, K., Klatzmann, D., Cole, S., Danos, O., Retzel, E., Tiollas, P., Haase, A. and Wain-Hobson, S. (1985) Nucleotide sequence of the visna lentivirus-relation to the AIDS virus. Cell 42, 369-382. Spira, T.J., Bozeman, L.H., Holman, R.C., Warfield, D.T., Phillips, S.K. and Feorino, P.M. (1987) Micromethod for assaying reverse transcriptase of human T-cell lymphotropic virus type III/ lymphadenopathy-associated virus. J. Clin. Microbial. 25, 97-99. Stone, L.B., Scolnick, E., Takemoto, K.K. and Aaronson, S.A. (1971) Visna virus: a slow virus with an RNA dependent DNA polymerase. Nature, 229, 257-258.
129
VIRMET 1277
Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester David R. Sargan,
Neil J. Watt and Ian McConnell
Department of Veterinary Pathology, University of Edinburgh, Edinburgh, U.K. (Accepted
13 September
1991)
Summary The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeticiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15~11of sample, is rapid, and is able to detect virus at titres below 103/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection. Reverse transcriptase;
Lentivirus; Radiolabelling; Cell harvester; Visna/maedi; HIV
Introduction Intensive research on retroviruses of the lentivirus subgroup has been triggered by the discovery of the human immunodeficiency viruses HIV I and II, the causative agents of the disease AIDS, and their identification as Correspondence to: D.R. Sargan, Dept. of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 lQH, U.K.
130
members of this subgroup (reviewed in Narayan and Clements, 1989.) Lentiviruses can be distinguished from most other mammalian retroviruses by the divalent cation and template specificities of their RNA-dependent DNA polymerases (reverse transcriptases) (Lin and Thormar, 1970; Goodman and Spiegelman, 1971; Stone et al., 1971; Hoffman et al., 1985). Assays for these enzymes can be a sensitive and rapid test for the presence of viruses in this group (Spira et al., 1987; Gregersen et al., 1988). We have investigated the characteristics of the reverse transcriptase of the sheep lentivirus visna/maedi virus (de Boer, 1975) in tissue culture media and cell lysates by using a microscale semi-automated assay. The assay measures the inco~oration of radiolabelled thymidine triphosphate into nucleic acids using an oligo(dT) primer and poly(rA) template. Incorporated nucleotides are distinguished from unincorporated using either acid precipitation (if protein concentration in the sample is low) or binding to cation exchange paper (if protein concentration in the sample is higher). The assay is performed in microtitre plates, similar to that reported by Gregersen et al. (1988) but unlike their assay, a cell harvester is used to filter and process assay supernatants, allowing handling of several hundred samples at once. We have used the assay to demonstrate directly the presence of viral reverse transcriptase within infected cells from permissive cultures within 36 h of visna infection in vitro. The assay cannot, however, cope with the very high protein concentrations (> 50 mg/ml) found in lymph and sera from infected animals. Both nonspecific RNase and DNase activities in samples may interfere with the assay, and we have developed protocols to minimise these effects. Materials and Methods Materials
E3H]TTP (20 C/1 mmol) and [32P]TTP (400 Ci/mmol) were from Amersham. Synthetic otigo- and polynucleotides and human placental ribonuclease inhibitor were from Pharmacia. DEAE paper was from Whatman. Glass fibre filters (45 micron pore size) were from Skatron AS. Tissue culture media and plasticware were from Gibco. Sheep DNA was prepared by standard methods from peripheral blood of Scottish Blackface x Finn sheep. All other reagents were of Analar or equivalent quality. Methods Visna virus samples
Visna virus was grown in primary cultures of choroid plexus cells (Weybridge cell line) between passages 15 and 30 in Dulbecco’s Minimal Essential Medium containing 1% foetal calf serum. Cells were infected at MOI below 0.05 with visna virus of strain 1514 (Sonigo et al., 1985) or one of two
131
British strains designated K184 and EVl (Sargan et al., 1991). Tissue culture supernatants were harvested from infected cells for use as sources of virus at various times. Larger cell debris was removed from these supernatants by centrifugation for 10 min at 5000 x g, and the clarified virus containing media either used directly in the assay or subjected to further purification by a modification of the method of Hoffmann et al., 1985. Virus was recovered from 12-ml samples of the clarified media by centrifugation for 40 min at 192 000 x g and resuspended in 250 ~1 TNE (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA). Pooled samples were layered onto a 3-ml 15% sucrose cushion in TNE above a l-ml pad of 65% sucrose in TNE and spun at 100 000 x g for 1 h. A 0.5-ml sample from the interface of the 2 sucrose solutions was then diluted to 2 ml and loaded onto a linear lo-ml 2065% sucrose gradient in TNE. This was subjected to centrifugation for 16 h at 192 000 x g and 20 drop fractions were collected by upward displacement for reverse transcriptase assay. In vitro infected choroid plexus cells for assay of cell-associated RT activity were stripped from culture vessels by scraping and washed twice with Hanks Balanced Salt Solution (HBSS) containing 1% foetal calf serum and twice in HBSS alone. They were resuspended at 5 x lo6 cells/ml in the same buffer. 0.5% Triton X-100 was added and the cells lysed by homogenization. Cells were then diluted to 1 x 106/ml using HBSS prior to assay for reverse transcriptase. Primary cultures of synovial libroblasts from CAEV-infected animals were maintained in culture for 10 days prior to treatment as described above. Reverse transcriptase assay Samples for assay (15 ~1) were placed in wells of 96-well U-bottomed ELISA plates. 10 ul of 2.5 x reaction mix (2.5 x reaction mix = 375 mM KCl, 25 mM MgC12,25 mM Tris-HCl, pH 8,5 mM DTT, 1.25 mM EGTA, 0.25% Triton X100, 62.5 ug/ml BSA, 75 ug/ml poly(rA).oligo(dTiz_is), 5% (v/v) ethane diol (deionized), 250 uCi/ml (or as stated in text) [3H]TTP (concentration 12,.5 uM in all cases). A 5 x reaction mix stock without template or primer and DTT was routinely stored at - 20°C for periods of several weeks. The plate was then incubated for 1 h at 37°C or as stated in the text. The assay was stopped and harvested by one of two methods: (I) TCA precipitation assay 100 ul of 10% (w/v) trichloroacetic acid (TCA) containing 50 ug/ml yeast RNA (Sigma) was added to each well and the plate left on ice for 15 min. Samples were then harvested onto a glass fibre mat filter (Skatron) using a Titertek cell harvester. Wells and filters were washed 3 x with 5% (w/v) TCA, 30 (w/v) sodium pyrophosphate and 2 x with 70% ethanol and dried. Filter discs were cut out and counted in Optisafe (a proprietary scintillant, supplier LKB) in a beta counter. (2) DEAE-paper
assay
100 ul of 10 x TE (100 mM Tris-HCl,
pH 8, 10 mM
132
EDTA) were added to each well and samples were harvested onto Whatman DEAE paper (DE81) supported by a glass-fibre filter on the cell harvester. The wells and filter were sequentially washed three times with 10 x TE containing 0.1% Triton X-100 and twice with 10 x TE containing 0.15 M NaCl and 0.1% Triton X-100. Filters (both DEAE paper and glass-fibre backing) were dried and counted as before. This method proved to have similar sensitivity to method (1) above, but also lower backgrounds in the presence of protein (see Results). Samples for DNA gel electrophoresis were prepared using [32P]TTP in standard assays. After stopping the assay using 100 pl 10 x TE, assays were phenol chloroform extracted and ethanol precipitated, prior to electrophoresis on 8.3 M urea polyacrylamide gels by standard methods. Results Characteristics of the reverse transcriptase of visna virus; salt optima and detergent characteristics
Using the microscale assay and the cell harvester we measured incorporation of [3H]TTP into acid insoluble products by purified visna virus samples under a number of different conditions. Parameters investigated included KC1 and Mg2+ concentrations, template concentration and the ethane diol, EGTA and Triton X-100 dependence of the assay. In all of these assays a 60-min incubation at 37°C of 15 ~1 of a 20 or loo-fold dilution of sucrose gradient purified visna type 1514 from infected WSCP cells was used in a 25 l.d assay. No RNase inhibitor or carrier DNA was used. The results of these assays are shown in Fig. 1. As can be seen the assay has a broad optimum for Mg2+ concentration around 7-10 mM, and an optimum for KC1 of 150 mM. Optimum Mn2+ concentration in the assay is 0.6 mM to 0.8 mM, but this divalent cation gives much lower maximum incorporation than Mg2+ (Table TABLE 1
Template for assay
cpm in assay
None poly (rA).oligo poly (dA).oligo oligo (dTrz-1s) poly (rA).oligo poly (rA).oligo
1366+46 19678+770 1519&493 677 f 89 35844+6009 4 643 + 334
(dTrs_rs) (dTrs_rs) (dTr&, + 0.5 mM EGTA (dTrz_rs), + 0.8 mM Mn’+, no Mg2+
Sucrose gradient purified visna virus strain K184 was diluted 20 times in TNE, and duplicate samples were assayed using the TCA precipitation assay, in the presence of 75 ug/ml of each template as shown. Except where stated these assays were in 5 mM MgC12 without EGTA. Mean counts incorporated in a 60-min assay are shown.
0' 0
loo
200
300
400 KCI mM
0
2-5
5
7-5 MgCIz
10
mM
Fig. 1. Characteristics of the reverse transcriptase assay. Assays were performed on various samples of sucrose gradient puriiied visna virus type 1514, diluted 20 or 100 times with HBSS, using 2.5 @ci 13H]‘ITP per assay and reaction conditions as stated. (A) Effect of varying KC1 concentration; (B) effect of varying MgC& concentration; (C) effect of varying pH.
1). Maximum in~o~oration occurred at pH 8.25. In~o~oration was totally dependent on the presence of Triton X-100, but independent of its concentration in the range 0.05-0.5%. The assay was activated by EGTA (see below). Ethane dial had a slight activating effect. Incorporation in the assay was maximal at template concentrations above 10 ug/ml in these conditions, These results show that visna virus reverse transcriptase has very similar reaction characteristics to those already published for HIV (Hoffman et al., 1985; Rey et al., 1984; Cheng et al., 1987) The assay measures a reverse ~r~nscriF~ase activity
To control for the possibility that the assay was measuring DNA polymerase activity, a poly(dA) template was substituted for the poly(rA) template, again
134
using an oligo(dT) primer (Table 1). Little activity was seen using this template. Similarly little activity was seen in the absence of template but in the presence of primer, suggesting that no terminal transferase activity was being detected by the assay. Similar results were found when clarified tissue culture supernatant from visna infected cells or lysed infected cells were used.
Sensitivity
and time course of the assay
TABLE 2
Gross cpm in assay Sample dilution
Cell harvester
Filtration
l/l l/3 l/9 l/21 Mock-infected,
6 218 *438 4 340 + 251 1548+116 855& 146 110+26
6613k530 4 409 + 290 3 631+253 2154k156 1218
l/l
manifold
Tissue culture supernatants from visna virus type EVl infected WSCP cells (titre = 3.8 x 10’ TCIDSo/ml-‘) were diluted as shown with HBSS and assayed (0.5 pCi [3H]TTP, 60 min). Assays were filtered using either the Flow cell harvester or a Millipore filtration manifold, and filters washed extensively using in both cases 5% TCA, 3% sodium pyrophosphate followed by 70% ethanol.
1
2
3
4
18h
. Time
Fig. 2. Time course of incorporation in the assay. Visna type 1514 from various sources was incubated in triplicate assays with 13H]TTP and the assay stopped at various times. Incorporation into acid-soluble material was measured. (0) Clarified tissue culture medium, no RNase inhibitor; (a) clarified tissue culture medium, 0.25 U/assay RNase inhibitor; (A) sucrose gradient purified virus diluted 1:lOO with HBSS.
NO _
Ca2+
II-L-!3 to 30 100
Fig. 3. (A) Sensitivity of the reverse transcriptase assay. Titres of different strains of virus in tissue culture media were measured by serial dilution and plating onto choroid plexus cells. The same samples were used in reverse transcriptase assays, using 2.5 $i [3H]‘ITP and reaction conditions as stated. Harvesting was by acid precipitation. Points show mean inco~ration in triplicate assays. Error bars are omitted for clarity. (A) Visna strain K184; (Cl) a British CAEV isolate; (e) visna 221050; (0) visna strain 1514; (II) visna strain EVl. (B) Sizes of reverse transcriptase products in the assay. Sucrose gradient purified visna virus strain 1514 was diluted by the amounts shown with HBSS and used in 2-h assays with [s2P]TTP (2.5 @i/assay, 100 Ci/mmol, 12.5 ul assays). Products of the assay were extracted with phenoi/chlorofo~ denatured and sized on 7.5% ~lyac~l~ide 8.3 M urea gels. Lanes l-4: assay in the presence of 0.5 mM EGTA, no Car-+; lanes 5-8: as l-4, but 2.0 mM Ca2+. Sizes of marker DNA fragments are shown.
To validate the use of the cell harvester to filter assays, duplicate assays were performed on visna-infected tissue culture supernatants which were harvested using either the cell harvester or a Millipore barrel filter apparatus and Whatman GF/C type filters. Incorporation as measured by either assay was very similar (Table 2), but background as measured on the cell harvester was much lower. Sucrose gradient purified visna virus (strain 1514) or clarified tissue culture
136
supernatants containing virus were used in assays to investigate the effect of different incubation periods. When no RNase inhibitor was present incorporation into acid precipitable material plateaued after 60 min in tissue culture supernatants. However, when purified virus samples were used, incorporation remained linear for 4 h and continued to increase overnight (Fig. 2). Addition of RNase inhibitor to the assay increased incorporation of TTP at later times when clarified tissue culture media were used, but had little effect on purified samples. The sensitivity of the assay was measured by titrating virus present in tissue culture media using HBSS. The titre of infectious virus in the samples was measured by examining the dilutions for ability to induce CPE on WSCP in 96 well plates. Such assays were performed for visna strains 1514 and 221050, and the 2 British visna isolates (K184 and EVI), as well as for a British CAEV isolate (Fig. 3A). Reverse transcriptase activity was measured in 60-min assays at 37°C containing 2.5 PCi [3H]TTP, but in the absence of RNase inhibitor. The sensitivity of the assay varied with strain, the highest sensitivity being found with visna K184. For this virus a strong positive signal (20000 cpm above background) was found to correspond to the presence of lo3 virus TCIDSO/ml with a sample size of 15 ~1. (Note, however that the proportion of defective virions in the assay is unknown.) Sensitivity is improved approx. lo-fold by using [32P]TTP (10 ~Ci~s~ple, 50 Ci/mmol) in a 4-h assay in the presence of placental RNase inhibitor (data not shown). It was noted that the assay was not linear with dilution of the virus. A possible explanation for this observation emerged when the size of transcripts produced in the reaction was measured by electrophoresis (Fig. 3B). When higher dilutions of virus samples were used, transcript sizes increased. These effects were particularly marked in virus samples from tissue cultures in which cells had been lysed by the virus (data not shown). Competition for template or for free nucleotide triphosphates is not likely to cause these results because template concentration could be reduced 2.5-fold without affecting the level of incorporation into the assay (data not shown) and total incorporation was less than 4% of supplied nucleotide t~phosphates in any sample. Because the assay is linear with time when purified virus is used, it is unlikely that viral RNase H activity destroying the reaction template is the cause of non-linearity in nonpurified samples, but possible that nonspecific deoxyribonuclease and ribonucleases are present in these samples, which at high concentrations destroyed the reaction products. When EGTA was omitted from the assay the size of synthesized transcripts decreased, and it was possible to increase the linearity of the assay at low dilutions of virus by dialysis of virus samples against TNE. These observations are consistent with the presence of a calciumdependent deoxyribonuclease similar to many of those purified from eukaryotic cells (Duerksen and Connor, 1978). We therefore added 100 pg/ ml of purified DNA from sheep thymus to each reaction as a carrier. In some, though not all samples, this increased the linearity of the reaction with respect to dilution of the virus sample, and the total counts inco~orated in the
137
reaction at high virus concentrations. In contrast, addition of Ca*+ to the assay reduced transcript sizes (Fig. 3B). Measurements
of reverse transcriptase
in infected cells
We have attempted to measure reverse transcriptase activity in visna-infected cell populations and body fluids from visna and CAEV-infected animals. Harvesting of the assay using TCA precipitation and filtration onto glass-fibre filters led to unacceptably high backgrounds with these materials. We therefore developed an alternative means of harvesting the assay. The assay was stopped with 100 ul 10 x TE and harvested onto DEAE paper backed by a glass-fibre filter for strength, using the cell harvester as before. Sequential washes with salt solutions were then used to remove unincorporated nucleotides. After some experimentation it was found that two washes with 10 x TE, 0.1% Triton X100, followed by two washes with 0.15 M NaCl, 10 x TE, produced high signals combined with relatively low background. This assay allowed detection of reverse transcriptase in extensively washed WSCP cells at short times (36 h) after infection in vitro (MO1 = 0.1-1.0) (Table 3). This was considerably before these cells showed any cytopathic effect, though after the first appearance of viral transcripts (Sargan et al., in preparation). The assay also allowed detection of cell-associated reverse transcriptase in primary cultures of synovial libroblasts from CAEV-infected goats. Though the DEAE paperbased assay allowed detection of cell-associated virus, it was still unable to cope TABLE 3 Reverse transcriptase
assays on cell lysates
Sample source
Visna Visna Visna Visna Visna Visna Visna Visna CAEV CAEV
1514 from WSCP, TC fluid (TCA) 1514 from WSCP, TC fluid (DEAE) 1514 in WSCP cell lysates, 8 h p.i. 1514 in WSCP cell lysates, 24 h p.i. 1514 in WSCP cell lysates, 36 h p.i. 1514 in WSCP cell lysates, 48 h p.i. 1514 in WSCP cell lysates, 5 days p.i. 1514 in WSCP cell lysates, 5 days p.i. (TCA) goat synovial membrane (expt. 1) goat synovial membrane (expt. 2)
cpm in assay Virus-positive sample or infected animal
Control sample or uninfected animal
2 202 f 384 3 136k50 202k25 209k9 462 f 58 922 f 53 20 490 + 2 960 21414k3653 1057+66 3 596 + 293
290+61 106+48 211+39 NA NA NA 387 f 209 1127k340 184+35 NA
Rows 1 and 2: a comparison of TCA precipitation and DEAE paper filtration; rows 3-6: detection of cell-associated reverse transcriptase by DEAE paper filtration in WSCP cell populations infected in vitro at various times post infection; rows 7,8 detection of cell-associated reverse transcriptase in lysates of cell populations from CAEV-infected goats (2 independent experiments are shown). Synovial membrane cells from biopsy specimens were maintained in tissue culture for 10 days prior to assay. Cell populations were lysed and prepared for assays as stated. Assays using 1 uCi [ H]TTP were performed and harvested using the DEAE paper method (except rows 1 and 8). Figures represent mean counts incorporated, and standard errors of three samples.
138
with the high levels of protein found in lymph or synovial fluid from infected animals (data not shown). Discussion
A semi-automated and therefore rapid assay for lentivirus reverse transcriptase in fluid media or in lysed cell populations is presented here. The assay is fairly inexpensive to perform, the cost being largely determined by the amount of radioisotope used. Modification of the harvesting procedure in the assay by using DEAE paper and salt washes allows assay of samples with protein concentrations greater than 5 mg/ml, but with slight loss of sensitivity when compared with assay by TCA precipitation. The assay has not been able to detect lentivirus reverse transcriptase in direct measurements of lymph or serum samples, but can detect reverse transcriptase in extensively washed infected cells.
Visna
Present
Ccpel
+
+
+
+
+
+
Fig. 4. Autoradiograph of part of a filter mat from the cell harvester after assay of 6 visna virus positive and 6 negative samples. Tissue culture fluid from infected and uninfected cell cultures was used in 25 ul assays containing 0.5 uCi/assay [‘*P]TTP. After incubation, harvest and wash the filter mat was subject to autoradiography for 2 h at room temperature.
139
The amount of product given by the reaction and the reaction’s linearity with time and virus concentration are increased by the presence of RNase inhibitors and of carrier DNA. This suggests that contamination of viral samples with both DNases and RNases may be a common problem in reverse transcriptase assays. The effect of DNases in particular was increased in virus samples from cell cultures in which marked viral-induced cell lysis had occurred. The effect could also be reduced by titration of the sample into a calcium-free buffer. The assay is best suited to the quantification of virus in large numbers of samples of low protein concentration (for instance tissue culture media derived either from virus culture or from coculture of potentially infected cells with permissive cell lines). Very rapid screening for product formation can make use of [32P]TTP in the assay, and use autoradiographs of the dried filters to detect products of the reaction (Fig. 4). By this method single positives can be found in thousands of samples, and a single worker can easily handle several hundred samples in a day. Although previous reports have shown that the visna reverse transcriptase is Mg2+-dependent, no detailed characterization of the requirements of the enzyme has been presented (Lin and Thormar, 1970; Stone et al., 1971). The reaction optima for sheep lentiviruses and for HIV- 1, bovine immunodeticiency virus (BIV), and caprine arthritis encephalitis virus (CAEV) (in terms of salt concentration etc.) are rather similar (Kashanchi et al., 1991; Hoffman et al., 1985; Clements et al., 1980). (The enzymes have optima for KC1 of around 150 mM, for pH 8.0-8.25, and a preference for Mg2+ rather than Mn2+) An ancestral relationship between HIV-l and the lentivirus group in terms of genome structure and sequence has been previously demonstrated (Gonda et al., 1985; Chui et al., 1985; Sonigo et al., 1985; Querat et al., 1990). The assay may therefore have general application as a method for assessing the presence of lentiviruses in samples when the type of virus has not been fully characterized, or no antibody based test is available.
Acknowledgements
We wish to acknowledge excellent technical assistance from Mr I. Bennet, gifts of viral samples and other materials from Dr M. Dawson and Dr G. Harkiss and to many colleagues for helpful discussions. The research was supported by a project development award from the Wellcome Trust.
References Cheng, Y.-C., Dutschman, G.E., Bastow, K.F., Sargadharan, M.G. and Ting, R.Y.C. (1987) Human immunodeficiency virus reverse transcriptase: general properties and its interaction with nucleoside triphosphate analogs. J. Biol. Chem. 262, 2187-2189. Chui, I.-M., Yaniv, A., Dahlberg, J.E., Gazit, A., Skuntz, SF., Tronick, S.R. and Aaronson, S.A. (1985) Nucleotide sequence evidence for relationship of AIDS retrovirus to lentivirus. Nature 3 17, 366-368.
140 Clements, J.E., Narayan, 0. and Cork, L.C. (1980) Biochemical characterisation of the virus causing leukoencephalitis and arthritis in goats. J. Gen. Viral. 50, 423427. deBoer, G.F., Teopstra, C. and Howens, D.J. (1975) Studies in the epidemiology of maediivisna in sheep. Res. Vet. Sci. 26, 202-208. Duerksen, J.D. and Connor, K.W. (1978) Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases. Mol. Cell. Biochem. 19, 93-l 12. Gonda, M.A., Wong-Staal, F., Gallo, R.C., Clements, J.E., Narayan, 0. and Gilden, R.V. (1985) Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus. Science 227, 173-177. Goodman, NC. and Spiegelman, S. (1971) Distinguishing reverse transcriptase of an RNA tumor virus from other known DNA polymerases. Proc. Nat]. Acad. Sci. USA 68, 2203-2206. Gregersen, J.P., Wege, H., Preiss, L. and Jentsch, K.D. (1988) Detection of human immunodeficiency virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay. J. Virol. Methods 19, 161-168. Hall, J.G. and Morris, B. (1962) The output of cells in lymph from the popliteal node of sheep. Quart. J. Exp. Physiol. 47, 36&369. Hoffmann, A.D., Banapour, B. and Levy, J.A. (1985) Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147, 326335. Kashanchi, F., Lui, Z.Q., Atkinson, B. and Wood, C. (1991) Comparative evaluation of bovine immunodeticiency-like virus infection by reverse transcriptase and polymerase chain reaction. J. Viral. Methods 31, 197-210. Lin, F.H. and Thormar, H. (1970) Ribonucleic acid dependent deoxyribonucleic acid polymerase in visna virus. J. Virol. 6, 702-704. Narayan, 0. and Clements, J.E. (1989) Biology and Pathogenesis of Lentiviruses. J. Gen. Virol. 70, 1617-1639. Querat, G., Audoly, G., Sonigo, P. and Vigne, R. (1990) Nucleotide sequence analysis of SAOMVV, a visna -related ovine lentivirus: phylogenetic history of lentiviruses. Virology 175,434 447. Rey, M.A., Spire, B., Dormont, D., Barre Sinoussi, F., Montagnier, L. and Clermann, J.C. (1984) Characterization of the RNA-dependent DNA polymerases of a new human T lymphotropic retrovirus (lymphadenopathy associated virus). Biochem. Biophys. Res. Comm. 121, 126133. Sargan, D.R., Bennet, I.D., Cousens, C., Roy, D.J., Blacklaws, B.A., Dalziel, R.G., Watt, N.J. and McConnell, I. (1991) Nucleotide sequence of EVI, a British isolate of maedi-visna virus. J. Gen. Virol. 72, 1893-1903. Sonigo, P., Alizon, M., Staskus, K., Klatzmann, D., Cole, S., Danos, O., Retzel, E., Tiollas, P., Haase, A. and Wain-Hobson, S. (1985) Nucleotide sequence of the visna lentivirus-relation to the AIDS virus. Cell 42, 369-382. Spira, T.J., Bozeman, L.H., Holman, R.C., Warfield, D.T., Phillips, S.K. and Feorino, P.M. (1987) Micromethod for assaying reverse transcriptase of human T-cell lymphotropic virus type III/ lymphadenopathy-associated virus. J. Clin. Microbial. 25, 97-99. Stone, L.B., Scolnick, E., Takemoto, K.K. and Aaronson, S.A. (1971) Visna virus: a slow virus with an RNA dependent DNA polymerase. Nature, 229, 257-258.
