Human T-cell leukaemia virus type I is highly sensitive to UV-C light

Akira Shimizu1,2, Nobuaki Shimizu1, Atsushi Tanaka1, Atsushi Jinno-Oue1, Bibhuti Bhusan Roy1, Masahiko Shinagawa1, Osamu Ishikawa2 and Hiroo Hoshino1

1 Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
2 Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan

Correspondence
Hiroo Hoshino
hoshino{at}med.gunma-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The biological characteristics of human T-cell leukaemia virus type I (HTLV-I) are not yet well understood. UV light C (UV-C) sensitivity of HTLV-I was studied using a newly established infectivity assay: infection with cell-free HTLV-I dose-dependently induced syncytial plaques in cat cells transduced with the tax1 gene of HTLV-I. HTLV-I was inactivated by a much lower UV dose than bovine leukaemia virus (BLV). The D10 (10 % survival dose) of HTLV-I was about 20 J m–2, while that of BLV was about 180 J m–2, which was similar to the reported D10 of BLV. The UV sensitivity of HTLV-I and BLV was also examined by detecting viral DNA synthesis 24 h after infection. The D10 values determined by PCR using the gag primers for HTLV-I and BLV were close to those determined by the infectivity assays. Further PCR analyses were then performed to determine D10 values using several different primers located between the 5'-long terminal repeat (5'-LTR) and the tax1 gene. The difference in UV sensitivity between HTLV-I and BLV was detected very early during replication, even during reverse transcription of the 5'-LTR of irradiated viruses, and became more prominent as reverse transcription proceeded towards the tax1 gene. Chimeric mouse retroviruses that contain the LTR-tax1 fragments of HTLV-I and BLV were made and hardly any difference in UV sensitivity was detected between them, suggesting that the difference was not determined by the linear RNA sequences of HTLV-I and BLV. HTLV-I was found to be much more sensitive than other retroviruses to UV.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human T-cell leukaemia virus type I (HTLV-I) was the first retrovirus discovered related to human diseases. It is the aetiological agent of adult T-cell leukaemia (Hinuma et al., 1981; Poiesz et al., 1980) and of HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Jacobson et al., 1988; Osame et al., 1986). The mechanism of entry of HTLV-I into cells or the reactivity of HTLV-I with human sera has frequently been investigated by using the vesicular stomatitis virus (VSV) pseudotype assay (Clapham et al., 1984; Hoshino et al., 1985), or by HTLV-I-specific PCR using concentrated HTLV-I (Fan et al., 1992). As for the infectivity of HTLV-I, it has mainly been assessed by evaluating syncytium formation, in which HTLV-I-producing cells and non-infected cells are co-cultured (Hoshino et al., 1983; Nagy et al., 1983). However, virions of HTLV-I themselves, or cell-free HTLV-I, have rarely been used for examination of the infection mechanism. We have recently developed a new system by which we could detect syncytial plaque formation after infection of Tax1-expressing feline 8C cells with cell-free HTLV-I. In the present study, we examined the UV light C (UV-C) sensitivity of HTLV-I by this method. As we previously reported, the PCR system efficiently detects reverse-transcribed viral DNA in target cells after infection of cell-free HTLV-I (Haraguchi et al., 1994). The UV sensitivity of HTLV-I was also investigated using this PCR system.

It has been known for a long time that retroviruses are, in general, more UV-resistant than other RNA viruses containing genomes of a similar length (Henderson et al., 1992; Levinson & Rubin, 1966; Murphy & Gordon, 1981; Owada et al., 1976). As for the dose required to reduce the infectivity to 10 % (D10), it has been reported that the D10 values of murine leukaemia virus (MuLV), bovine leukaemia virus (BLV) and human immunodeficiency virus type 1 (HIV-1) are 370, 220 and 280 J m–2, respectively (Guillemain et al., 1981; Yoshikura, 1989). Unexpectedly, we found that the D10 of HTLV-I was as low as about 20 J m–2 and that the reverse transcription of HTLV-I was a highly UV-sensitive stage. We investigated and discuss possible mechanisms that could explain the high UV sensitivity of HTLV-I.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
MT-2 (Miyoshi et al., 1981), 8C/HTLV-IMEL5, 8C/HTLV-I2M and PG-4/HTLV-IMT-2 cells were used as HTLV-I-producing cells in this study. The 8C/HTLV-IMEL5 and 8C/HTLV-I2M cells are 8C cells (Fischinger et al., 1973) derived from feline kidney cells persistently infected with HTLV-IMEL5 (Yanagihara et al., 1991) and HTLV-I2M derived from a Melanesian and a Japanese patient, respectively. PG-4/HTLV-IMT-2 cells were persistently infected with HTLV-IMT-2 derived from a Japanese patient and were newly established by co-culturing an HTLV-IMT-2-producing rabbit T-cell line, Ra-1 (Miyoshi et al., 1983), with a feline glial cell line, PG-4 (Haapala et al., 1985). The 8C/HTLV-I2M line c77 was a subclone of 8C cells that were co-cultivated with lethally irradiated ATL-2M cells producing HTLV-I2M (Hoshino et al., 1983). The BLV-producing cells were fetal lamb kidney (FLK) cells (van der Maaten & Miller, 1975). The indicator cells for infection with cell-free HTLV-I or BLV were 8C and 8C/Tax1. The 8C/Tax1 cells were derived from 8C cells transduced with the tax1 gene of HTLV-I. When 8C cells had been transduced with a retroviral Tax-1-expression vector, DGL-Tax1 (Akagi et al., 1991), they formed many syncytia after infection with HTLV-I produced by 8C/HTLV-I or PG-4/HTLV-I cells. Syncytium formation was inhibited by treatment with anti-HTLV-I seropositive human sera, a mouse mAb against HTLV-I gp46 or 3'-azido-3'-deoxythymidine (unpublished data). Details of this cell line will be described elsewhere. The indicator cells used for infection with chimeric Moloney murine leukaemia viruses (MoMuLVs) harbouring HTLV-I or BLV genome fragments were N4R (NP-2–CD4–ecoR). This is a cell line derived from NP-2 human glioma cells transduced with CD4 as well as an ecotropic MuLV receptor (Soda et al., 1999). The packaging cells used for chimeric virus production were Phoenix-E cells (http://www.stanford.edu/group/nolan/). The 8C, 8C/Tax1, 8C/HTLV-IMEL5, FLK and N4R cells were maintained in Eagle's minimum essential medium (E-MEM) containing 10 % fetal calf serum (FCS). Phoenix-E cells were maintained in Dulbecco's modified MEM (D-MEM) containing 10 % FCS. MT-2 cells were maintained in RPMI 1640 medium containing 10 % FCS. The PG-4/HTLV-IMT-2 cells were maintained in McCOY'S 5A medium containing 15 % FCS. All cells were maintained at 37 °C in a humidified, 5 % CO2 atmosphere.

Virus preparation.
To prepare cell-free virus samples, 8C/HTLV-IMEL5, 8C/HTLV-I2M, PG-4/HTLV-IMT-2, MT-2 and FLK cells were seeded at 6·25x105 ml–1 and incubated for 2 days. Cells and debris were removed from the culture supernatants by low-speed centrifugation, and the supernatants were passed through 0·45 µm filters. The filtrates were dispensed into small aliquots and stored at –110 °C until use.

UV irradiation and dosimetry.
Aliquots of 700 µl of supernatants that contained one of these virus samples was placed in the centre of a 35 mm plastic culture plate, kept on ice to eliminate thermal effects and gently agitated to ensure uniform irradiation. UV irradiation was carried out under a low-pressure mercury vapour lamp. The incident UV dose rate was determined using a spectroline DRC-100X dosimeter (Spectronics).

Determination of the UV sensitivities of HTLV-I and BLV.
The 8C/Tax1 cells recently established by us were used for the infectivity assay. The 8C/Tax1 cells were seeded in 2 ml medium in flat-bottomed 6-well plates at 2·5x104 cells ml–1. The following day, after irradiation of the viruses with UV light, the cells were infected with 300 µl of HTLV-I or BLV preparations at 37 °C for 1 h in duplicate. The inocula were removed, the cells were washed once with culture medium and then fresh medium was added to the plates. The culture medium was changed at 3 and 6 days. The next day, the cells were fixed with methanol and stained with Giemsa solution and syncytial plaque were counted using an inverted microscope. Cells that contained more than four nuclei were considered to be syncytia and lesions that consisted of three or more syncytia were judged to be plaques. Syncytium numbers in control cultures were about 200 per well for each experiment. Variation of experimental data for each point was within 10 % of corresponding mean value.

UV sensitivity of VSV.
8C cells were seeded into 35 mm plastic culture plates at 6·0x105 cells ml–1. The following day, VSV was inactivated with UV light and the cells were infected with 300 µl of VSV preparations at 37 °C for 1 h in duplicate. The cells were washed once with culture medium and medium containing agar was overlaid onto the plates. The next day, cells were stained with neutral red and numbers of plaques were counted using an inverted microscope.

UV sensitivity of cells.
8C, 8C/Tax1 or HeLa cells were seeded in 4 ml medium in 35 mm plastic culture plates at 500 cells ml–1. The following day, after removal of the medium and irradiation of the cells with UV light at 0–12 J m–2, fresh medium was added to the wells. After 7 days, cell colonies were counted.

UV sensitivities of HTLV-I and BLV determined by PCR.
8C cells were seeded in 1 ml medium in flat-bottomed 12-well plates at 2x104 cells ml–1. Each virus was irradiated with UV. Target 8C cells were then infected with viruses at 37 °C for 1 h, washed with PBS once and incubated for 24 h as described previously (Haraguchi et al., 1994; Yang et al., 1994). The m.o.i. was about 1 for each virus. Cells were lysed with buffer that contained 10 mM Tris/HCl (pH 8·0), 1 mM EDTA, 0·45 % NP-40 (Sigma), 0·45 % Tween 20 (Sigma) and 20 mg protease K ml–1 (Sigma), incubated at 52 °C for 2 h and heated at 96 °C for 10 min to inactivate the protease K. To detect viral DNA by PCR, 15 µl reaction mixture that contained 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 1·5 mM MgCl2, each dNTP at 2 mM, 60 ng sense and antisense PCR primers and Taq DNA polymerase (Roche) was added to 5 µl cell lysate. PCR using the various primers shown in Figs 3(a) and 5(a) was performed in a thermal cycler (Perkin-Elmer Cetus). The PCR cycle was repeated 25–37 times under the following conditions: denaturation at 94 °C for 1 min, annealing at 55 °C for 45 s and extension at 72 °C for 1 min. Amplified products were then separated by electrophoresis in an agarose gel containing ethidium bromide as described elsewhere (Haraguchi et al., 1994). We checked contamination of proviral DNA in virus inocula by PCR. No band was detected when PCR was done soon after inoculation of HTLV-I or BLV, indicating that neither proviral DNA in viral stocks nor strong stop DNA in virus stocks affected the results obtained. In addition to UV-irradiated viruses, cells were infected with serially diluted virus samples. We estimated the D10 by comparing the intensities of the PCR bands obtained with the UV-irradiated viruses with those bands obtained with diluted viruses, especially those obtained with the 1/10 dilution. That is, the intensity of each band was determined by densitometry and plotted on a semi-log graph and D10 values were determined using these graphs. D10 values determined using different PCR primers for a virus are expected to be dependent on the target size of UV irradiation. There should be an inverse proportional relationship between the D10 values and the target sizes of the virus genome examined, if the virus genome is homogeneously sensitive to UV. That is, the formula, D10xtarget size (bp)=constant is expected to be pertinent to UV-inactivation of each virus. Expected data are shown using dotted lines in Figs 4 and 6.



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Fig. 3. Determination of D10 values using different PCR primers. (a)–(b) Locations of the PCR primers used in this experiment. (a) HTLV-I and (b) BLV sequence-specific primers. For the primers, H and B and N and R mean HTLV-I and BLV and sense and antisense primer, respectively. A 1000 bp scale bar is shown. (c)–(f) Detection of viral DNA formation after infection of UV-irradiated viruses. 8C cells were infected with diluted (c–d) or UV-irradiated (e–f) HTLV-IMEL5 and BLV and cultured for 24 h. Viral DNA was detected by PCR (30 cycles) using the gag primers in Fig. 3(a). The five lanes of (c)–(d) were obtained using virus dilutions from 1/1 to 1/20. The six lanes of (e)–(f) were obtained using virus irradiated at dose from 0 to 400 J m–2 for HTLV-IMEL5 and from 0 to 540 J m–2 for BLV. (g)–(h) Densitometry of PCR bands. PCR bands were subjected to densitometry and their intensities are shown using arbitrary units. Open arrows show the intensities that the 1/10 diluted viruses gave upon PCR and closed arrows show D10 values. D10 values for HTLV-IMEL5 and BLV were estimated to be 25 and 135 J m–2, respectively.

 


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Fig. 5. UV sensitivity of the early stages of reverse transcription. PCR primers used in this experiment. (a) For HTLV-I, we used the antisense primer H-U5-R and four sense primers to examine PCR products of different lengths. The polypurine tract (PPT) is located in the tax1 gene before the U3 gene. (b) For BLV, we used the antisense primer B-U5-R and two sense primers. A 100 bp scale bar is shown. (c)–(d) Detection of viral DNA formation after infection of UV-irradiated viruses. 8C cells were infected with diluted or UV-irradiated HTLV-IMEL5 (c) and BLV (d) and cultured for 24 h. Viral DNA was detected by using PCR (HTLV-I, 37 cycles; BLV, 33 cycles) using the HTLV-I primers H-pX2-N and H-U5-R and the BLV primers B-pX2-N and B-U5-R. PCR fragment sizes were calculated to be 823 bp. The left five lanes were obtained using virus dilutions from 1/1 to 1/20. The right six lanes are irradiation doses, from 0 to 400 J m–2 for HTLV-IMEL5 and from 0 to 540 J m–2 for BLV. Densitometry of the PCR bands. The open arrows show the intensities that 1/10 diluted viruses gave and the closed arrows show D10 values. D10 values for HTLV-I and BLV were estimated to be 150 and 360 J m–2, respectively.

 


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Fig. 4. D10 values determined by using various PCR primer pairs. The horizontal axis shows the distance from the beginning of reverse transcription (target size of UV irradiation) and the locations of the primers used. Experiments were repeated at least twice. Mean D10 values were plotted using closed symbols. The first-strand transfer (jump) occurs in the R region (between U5 and tax1). An apparent difference in the D10 values between HTLV-I and BLV was even detected at an early stage of reverse transcription. The D10 values for the tax1 and gag regions were calculated, shown by open symbols, on the assumption that there is an inverse relationship between the distance of PCR primer locations in the virus genomes from the start of reverse transcription and the D10 values determined by using the corresponding PCR primers. These calculated values were plotted using open symbols with dotted lines. Symbols: {bullet} and {circ}, HTLV-I; {blacksquare} and {square}, BLV.

 


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Fig. 6. D10 values determined using the PCR primers shown above. The horizontal axis shows the distance from the start of reverse transcription. Experiments were repeated at least twice and mean D10 values were plotted. Open symbols with dotted lines represent D10 values calculated based on the assumptions described in the legend of Fig. 4 and in the text: HTLV-I but not BLV gave values near the expected values. Symbols: {bullet} and {circ}, HTLV-I; {blacksquare} and {square}, BLV.

 
PCR primers for HTLV-I and BLV.
Oligonucleotide primers were synthesized (Hokkaido System Science) to detect reverse-transcribed DNA of HTLV-I and BLV. The names of the PCR primers, nucleotide sequences and orientation of the primers were as follows:

H-R-N, 5'-CGTCCGCCGTCTAGGTAAGTT-3'; H-U5-R, 5'-TGTGTTCTATGTTTCTCTCC-3'; H-gag-N, 5'-GGAGCCTTACCACACCTTCG-3'; H-gag-R, 5'-AGGCTGGACGACTAACACCTT-3'; H-pX1-N, 5'-CCACCCAGAGAACCTCTAAA-3'; H-pX2-N, 5'-TTGCATCTCCCCTTCGAAGA-3'; H-pX-R, 5'-GGGTGTACAGGTTTTGGGGC-3'; H-U3L-N, 5'-TGATACTGACTATGGGCC-3'; H-U3R-N, 5'-AGCGTGGAGACAGTTCAGGA-3'; B-R-N, 5'-CTTCTCCTGAGACCCTCGTG-3'; B-U5-R, 5'-TGTTTGCCGGTCTCTCCTGG-3'; B-gag-N, 5'-AACGGGTACCCTAACCCAACAA-3'; B-gag-R, 5'-GGGTTCCTTAGGACTCCGTC-3'; B-pX1-N, 5'-GGGATCCATTACCTGATAA-3'; B-pX2-N, 5'-TTCCCCGGGACTCCAATGAA-3' and B-pX-R, 5'-TGGGTCTCGCAGGTGAGCGT-3'. The first H and B mean HTLV-I and BLV and the last N and R mean sense and antisense, respectively. The primer locations in the HTLV-I and BLV genomes are shown in Figs 3(a) and 5(a).

Construction of chimeric MoMuLVs.
We constructed chimeric MoMuLVs that contain HTLV-I and BLV sequences as follows: fragments of HTLV-I or BLV were inserted into a MoMuLV genome vector containing the green fluorescent protein (GFP) gene after the pol region, pMX-GFP. When cells were infected with MoMuLVs containing the gene for GFP near the 3' end of their genomes, GFP was expressed in the cells. That is, DNA fragments containing the pX(PPT)-U5 regions, i.e. from the polypurine tract (PPT) to U5 (823 bp) of HTLV-I and BLV, were obtained by PCR using the primer pairs H-pX2-N and H-U5-R, and B-pX2-N and B-U5-R, respectively. The fragments were cloned into the NotI site of the expression plasmid pMX-GFP to obtain the plasmids pMX-GFP-HTLV-I and pMX-GFP-BLV. They were transfected into Phoenix-E packaging cells for the production of recombinant ecotropic MoMuLVs and recombinant viruses were harvested. Recombinant viruses were irradiated with UV light as described above and N4R human cells expressing ecotropic MoMuLV receptors were infected with these viruses to detect GFP-expressing cells by flow cytometry (Cyto Ace-100).

UV sensitivities of chimeric pX(PPT)-U5 MoMuLVs.
N4R cells were seeded at 2·5x104 cells ml–1. The next day, cells were infected with chimeric viruses in the presence of polybrene. Cells were incubated for 4 days and detached with trypsin and the infection level was determined by flow cytometry. About 30 % of cells were judged to be positive for GFP after infection of the non-irradiated chimeric viruses.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infectivities of HTLV-I and BLV after UV irradiation
We determined the UV sensitivity of HTLV-I and compared it with that of BLV as a control. HTLV-I or BLV samples were placed in 35 mm plastic culture dishes, irradiated with UV and used to infect 8C/Tax1 cells. After 7 days, syncytial plaques were counted and the D10 values of these two viruses were determined (Fig. 1a).



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Fig. 1. UV sensitivities of HTLV-I and VSV. (a) UV sensitivity of HTLV-I. HTLV-IMEL5 and BLV were irradiated with UV and infected into 8C/Tax1 cells, and syncytial plaques were counted after 7 days. Syncytial plaque formation (number of plaques) by non-irradiated viruses is taken as 100 % and the relative numbers of syncytial plaques (%) formed by irradiated viruses are shown. In control culture dishes for HTLV-I and BLV, about 200 plaques were formed. Variation of experimental data for each point was within 10 % of corresponding mean value. The dose of UV irradiation is shown on the horizontal axis. Symbols: {bullet}, HTLV-I; {blacksquare}, BLV. (b) UV sensitivity of VSV. VSV is irradiated with UV and infected into 8C cells. Plaques were counted the following day. Plaque formation by non-irradiated viruses (about 200 plaques per well) is taken as 100 % and the relative numbers of plaques (%) formed by irradiated viruses are shown. The vertical axis shows the relative number of plaques.

 
For HTLV-IMEL5, a Melanesian strain of HTLV-I, the number of syncytial plaques began to decrease at 1·5 J m–2 and was decreased to about half the number of plaques produced by the non-irradiated control at 5 J m–2. This strain was almost completely inactivated at 27 J m–2. For BLV, the number of syncytial plaques began to decrease at 45 J m–2 and was decreased to about half the number produced by the non-irradiated control at 70 J m–2. BLV was almost completely inactivated at 270 J m–2. The inactivation curve of HTLV-I decreased much more steeply with increasing UV dose than that of BLV (Fig. 1a). Thus, HTLV-I was markedly more sensitive than BLV to UV and a large number of syncytia were observed even after irradiation of BLV at 45 J m–2 (Fig. 2). The D10 of HTLV-I was estimated to be about 17 J m–2, while that of BLV was about 180 J m–2. This value for BLV is similar to that reported previously, 220 J m–2 (Guillemain et al., 1981), and those of other retroviruses (Guillemain et al., 1981; Levinson & Rubin, 1966; Yoshikura, 1989). We also examined the UV sensitivities of two Japanese strains, HTLV-IMT-2 and HTLV-I2M, produced by PG-4/HTLV-IMT-2 and 8C/HTLV-I2M cells, respectively, as described above and found that their D10 values were also as low as about 20–30 J m–2 (data not shown).



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Fig. 2. Syncytial plaque formation by HTLV-I and BLV. HTLV-IMEL5 and BLV were irradiated at 0 and 27 J m–2, and 0 and 45 J m–2, respectively, and used to infect 8C/Tax1 cells. HTLV-I was almost completely inactivated by irradiation of 27 J m–2, while BLV was not greatly inactivated even by 45 J m–2. Syncytia formed by HTLV-I were smaller than those formed by BLV.

 
UV sensitivity of VSV
We determined the UV sensitivity of VSV as a control. VSV samples were placed in 35 mm plastic culture dishes, irradiated with UV and infected into 8C cells. The next day, plaques were counted and the D10 of VSV was determined (Fig. 1b).

The number of plaques began to decrease at 2·3 J m–2 and almost completely inactivated at 14 J m–2. The D10 of VSV was estimated to be about 6 J m–2 (Fig. 1b).

UV sensitivity of cells
HTLV-I and VSV were so UV-sensitive that we examined the UV sensitivity of the cells as a control. 8C cells were seeded and were irradiated the following day with UV light. After 7 days, we counted viable cell colonies and determined the D10 for colony formation of the cells. Colony formation of the 8C cells was completely inhibited at 6 J m–2 and the D10 of the 8C cells was about 3 J m–2 (data not shown). We also used 8C/Tax1 or HeLa cells for this assay and obtained similar results: their D10 values were about 3–5 J m–2. Since these data were similar to those previously reported in normal lymphocytes and chronic lymphoid leukaemia lymphocytes (Bogdanov et al., 1997), we considered our determination of UV dose to be accurate.

Viral DNA formation after infection of cells with UV-irradiated viruses
We examined whether the difference in UV sensitivity between HTLV-I and BLV could be detected shortly after infection, i.e. at the stage of reverse transcription. 8C cells were infected with HTLV-IMEL5 or BLV irradiated with UV and cell lysates were made 24 h later and examined for the formation of viral DNA by using PCR and gag primers (Fig. 3a, b) that were located in the region about 7·5 kb from the start of reverse transcription. In addition to UV-irradiated viruses, serially diluted virus samples were also used to infect the cells to determine quantitatively the D10 by PCR: representative results are shown in Fig. 3(c–f). Fig. 3(c–d) shows the effect of virus dilution on the PCR assay, and Fig. 3(e–f) shows the result of infection with irradiated viruses. The D10 values of the two viruses were estimated by comparing the intensities of the PCR bands obtained with the UV-irradiated viruses with those of the bands obtained with 1/10 diluted viruses (Fig. 3g, h). The D10 values of HTLV-IMEL5 and BLV were estimated to be 25 and 135 J m–2, respectively. These data are similar to those determined with the infectivity assays (Fig. 1a). These findings revealed that the marked difference in UV sensitivity between HTLV-I and BLV was already detected at the reverse transcription step in which the negative strands of the virus genome are found.

We then used several PCR primer pairs located in different regions of the virus genome (Fig. 3a) and examined at which stage of reverse transcription a difference in D10 was initially detected. We plotted the D10 values determined by PCR using the tax1 and gag regions of the PCR primers on a semi-log graph (Fig. 4). The difference in UV sensitivity between HTLV-I and BLV was even detected using PCR primers located in the R-U5 region (Table 1). The curve for BLV declined gradually as reverse transcription proceeded, while that for HTLV-I declined steeply from the tax1 gene to the gag gene and to the end of reverse transcription of the viral RNA (Fig. 4). As for HTLV-I, we noticed that there seemed to be an inverse relationship between D10 and the distance of the region examined from the start of reverse transcription, as shown by the dotted line in Fig. 4.


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Table 1. Summary of the UV sensitivity of HTLV-I and BLV

HTLV-IMEL5 and BLV were irradiated with UV and their D10 values were determined by infectivity (syncytial plaque formation in 8C/Tax1 cells) and PCR assays (8C cells) as described in Methods. The D10 values of DNA formation determined using the gag PCR primers were similar to those determined by the infectivity assay. The target sizes of UV irradiation and their D10 values determined using HTLV-I but not BLV seemed to be in inverse proportion. The D10 values of BLV determined by PCR for R-U5 or tax1 were much smaller than expected from the results of the infectivity assay.

 
UV sensitivity of viral DNA formation in the early stages of reverse transcription
We further investigated the early stages of reverse transcription of the HTLV-I RNA genome more precisely, as there was a marked difference in UV sensitivity between HTLV-I and BLV even when R-U5 or tax1 primers were used (Fig. 4; Table 1). We used an antisense primer, H-U5-R, and four sense primers, H-R-N, H-U3R- N, H-U3L-N and H-pX2-N, corresponding to different positions to change the length of the amplified DNA (Fig. 5a, b): thus the expected PCR fragments were calculated to be 300, 423, 754 and 823 bp, respectively. Cells were infected with serially diluted virus samples in addition to UV-irradiated viruses to determine quantitatively the D10 by PCR (Fig. 5c, d) as described above. Irradiation at 150 J m–2 was estimated to be necessary to reduce the intensity of the PCR band for HTLV-I to 1/10 (D10) of the control level (Fig. 5c), while 360 J m–2 was necessary for BLV (Fig. 5d). An apparent difference in D10 values between HTLV-I and BLV was detected at the stage of reverse transcription of the U5-R region. After that, the D10 of HTLV-I was markedly decreased, while the D10 of BLV did not considerably change (Fig. 6). As for the D10 values of HTLV-I, determined by using the four different PCR primers, that is 365, 220, 200 and 150 J m–2, there again seemed to be an inverse relationship between these values and the target sizes of viral RNA detected by PCR. The D10 of BLV determined by the use of the B-pX2-N primer, shown by the solid line, was much higher than the predicted value, shown by the dotted line (Fig. 6), suggesting that the D10 for BLV was not proportional to the target size of viral RNA irradiated or that the UV-irradiated genome of BLV would be repaired during reverse transcription.

UV sensitivity of the pX(PPT)-U5 region of HTLV-I and BLV
Since a marked difference in the UV sensitivity of HTLV-I and BLV was apparent during reverse transcription of the U5-tax1 region (Figs 4 and 6), we investigated whether the difference in UV sensitivity between the HTLV-I and BLV genomes was due to differences in their RNA sequences in this region. Therefore, we constructed chimeric MoMuLVs (MoMuLV GFP-HTLV-I and MoMuLV GFP-BLV) by inserting an 823 bp sequence of HTLV-I or BLV, pX(PPT)-U5, into pMX-GFP to produce MoMuLV GFP chimeric viruses. In these viruses, most of the MoMuLV genome RNA would be reverse transcribed after HTLV-I and BLV pX(PPT)-U5 RNA.

Recombinant viruses were irradiated with UV and inoculated into N4R cells. Fluorescent cells were detected by flow cytometry 4 days after infection. The proportions of GFP-positive N4R cells infected with non-irradiated MoMuLV GFP, MoMuLV GFP-HTLV-I and MoMuLV GFP-BLV were 35, 23 and 20 %, respectively. Next, each virus was irradiated at 20, 100 and 500 J m–2 and used to infect N4R cells. The proportions of GFP-positive N4R cells were, 26, 17 and 0·9 % for MoMuLV GFP, 19, 13 and 1·0 % for MoMuLV GFP-HTLV-I and 16, 9 and 0·6 % for MoMuLV GFP-BLV, respectively, indicating that the three viruses were similarly inactivated in proportion to the irradiation dose. The D10 values of these three viruses were calculated to be about 420 J m–2 and were similar to the reported D10 of MoMuLV, 370 J m–2, determined by an infectivity assay (Guillemain et al., 1981). We concluded that no special UV-sensitive sequence was present in the 823 bp of the pX (PPT)-U5 regions of HTLV-I and BLV.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The UV sensitivity of RNA viruses has long been studied (Murphy & Gordon, 1981), but the mechanism of UV inactivation is still not clear. There have been several reports on the mechanisms of UV inactivation in retroviruses: retroviruses are much more resistant than other RNA viruses to UV, if the size of their RNA genomes is considered (Henderson et al., 1992; Levinson & Rubin 1966; Owada et al., 1976; Yoshikura, 1989). In our experiments, the D10 values for VSV and BLV determined by infectivity assays were found to be about 6 and 180 J m–2, in agreement with reported values, although their genomic RNAs are of a similar size. It is not yet known why retroviruses are so UV resistant. It has also not yet been determined which part of retrovirus virions or which step of the virus replication cycle is most sensitive to UV irradiation. HTLV-I and BLV each contain two pairs of single-stranded RNA genomes of a similar size, and they are closely related to each other among various retroviruses (Sagata et al., 1984). However, we found that there was a marked difference in UV sensitivity between these two viruses. The UV sensitivity of BLV or MoMuLV determined by us was similar to those previously reported for BLV (Guillemain et al., 1981) as well as for other retroviruses (Yoshikura, 1989). We showed here that there was nearly a 10-fold difference in the D10 values determined by the infectivity and the PCR assay, using the gag primer pairs between HTLV-I and BLV after the infection of irradiated cell-free HTLV-I and BLV. The sensitivity of HTLV-I to UV light was thus much higher than those of other retroviruses, and was accordingly similar to those of other RNA viruses.

There seems to have been no report describing the effect of UV irradiation on several phases of the progression of reverse transcription. Under our assay conditions for PCR, PCR bands would be detected once a negative-strand DNA is formed by reverse transcription. We showed that the D10 values determined by infectivity (syncytium formation assay) and viral DNA formation (PCR that will detect negative-strand DNA formation using the gag PCR primer pair) were similar (Table 1). This finding suggests that retroviruses are inactivated by UV because their negative-strand DNA is not completely synthesized.

As a target, putative UV-sensitive viral structures have already been mentioned in the literature. Lovinger et al. (1975) examined the UV sensitivity of reverse transcriptase and concluded that at least 5–10 % of the loss of infectivity in irradiated retrovirus preparations may be due to viral polymerase inactivation. Owada et al. (1976) examined the UV inactivation of avian sarcoma virus and concluded that inactivation is caused not only by RNA damage but also damage in RNA–protein linkage as well as polymerase inactivation. Henderson et al. (1992) studied HIV-1 and stated that the formation of dimers (uracil dimers) after UV-irradiation alone is not effective for virus inactivation, because reverse transcriptase has the ability to read through the damaged bases. The nucleotide, including the linear sequence as well as its secondary or tertiary structures, has been supposed to be a major target for UV inactivation. However, reports on RNA photobiology are very limited. It has been supposed that the formation of pyrimidine hydrates and cyclobutadipyrimidines is important in the UV inactivation of RNA viruses, as it is for DNA viruses (Murphy & Gordon, 1981).

There has not been an easy assay to quantify the infectivity of cell-free HTLV-I therefore, we used a new method that we established recently, where we could detect the formation of syncytia 1 week after infection of Tax-1-expressing 8C cells by cell-free HTLV-I. The D10 values of two cosmopolitan (Japanese) strains, 2M and MT-2, and one Melanesian strain, MEL-5, of HTLV-I were about 20 J m–2 by this method. The D10 of VSV determined using a plaque formation assay was 6 J m–2. Thus, HTLV-I was still more UV-resistant than VSV.

We thought of the possibility that, if an irradiated virus infects cells, which harbour part of the viral gene, the D10 obtained would be higher than the actual value. If so, we would not be able to determine an accurate D10 when we used target cells that have previously been transfected with a viral gene. Therefore, if we use 8C/Tax1 cells that contain the tax1 gene, the D10 of HTLV-I would be observed to be higher than the real D10 determined using cells without the tax1 gene. However, the D10 of HTLV-I in our experiments was still very low. Since the UV sensitivity determined by PCR using 8C cells not transfected with the tax1 gene (Figs 3–6) was similar to that determined by the infectivity assay using 8C/Tax1 cells, we concluded that the transfection of target cells by the tax1 gene did not greatly influence the results of UV inactivation of HTLV-I.

As shown in Figs 4 and 6, the difference in UV sensitivity between HTLV-I and BLV was already observed at a very early stage of reverse transcription or before formation of the strong-stop DNA. Thus, this stage of the reverse transcription of HTLV-I RNA was already very sensitive to UV. We constructed chimeric MoMuLVs that contained 823 bp fragments of the HTLV-I or BLV origins and tested their UV sensitivities. We could hardly detect any difference in UV sensitivity between the two chimeric MoMuLVs. This result suggests that the primary RNA sequence itself is not a determinant of the high UV sensitivity of HTLV-I.

It is expected that there will be an inverse relationship between the UV target size of RNA or DNA and the corresponding D10 when the UV target is homogeneously sensitive to damage induced by UV irradiation: (value of D10)x(target genome size)=constant. We examined whether this was true for HTLV-I and BLV. The D10 values determined by infectivity assays of HTLV-I and BLV were about 17 and 180 J m–2, respectively (Fig. 1a; Table 1); the target sizes of UV irradiation are about 8·5 kb. Thus, D10 values for the UV target size of 300 bp are calculated to be about 480 and 5100 J m–2 for HTLV-I and BLV, respectively, whereas the experimental values were 370 and 480 J m–2, respectively (Table 1). For HTLV-I, there was a good correlation between experimentally obtained values and calculated values while, for BLV, there was an apparent discrepancy, as shown in Figs 4 and 6. This finding suggests that HTLV-I is susceptible to UV damage according to its target RNA size, while UV-irradiated BLV genomes may not be damaged proportionally to their genome sizes.

Several possibilities to explain the difference in UV sensitivity between HTLV-I and BLV can be suggested: (i) the genomic RNA sequence of HTLV-I contains more UV-sensitive sequences, for example pyrimidine dimers, than that of BLV, (ii) the abilities of the reverse transcriptases of HTLV-I and BLV to read through UV-damaged lesions are different, (iii) the first-strand transfer (jump) step of HTLV-I is especially vulnerable to UV irradiation, (iv) the virion structures of HTLV-I and BLV are different, especially the nucleocapsid (NC) and RNA complex structures or (v) recombination between RNA genomes in virions takes place much more readily during reverse transcription of BLV than of HTLV-I.

The first possibility seems unlikely, as we showed that chimeric MoMuLVs harbouring the HTLV-I and BLV tax1-LTR regions of 823 bp were similarly resistant to UV irradiation. Their D10 values were about 420 J m–2, similar to that of the parental MoMuLV strain, suggesting that a factor(s) other than linear RNA sequence may be responsible for the difference in UV sensitivity. Even in the case of RNA, pyrimidine dimers have been thought to be a major target of UV irradiation (Murphy & Gordon, 1981). Therefore, we counted the pyrimidine dimers in HTLV-I and BLV as well as MoMuLV, and found that HTLV-I and BLV had quite similar patterns of pyrimidine dimer constitution (data not shown).

The second possibility also seems unlikely because UV-irradiated VSV RNA similarly functioned as a template to make cDNA using reverse transcriptase of either HTLV-I or BLV (data not shown). Figs 4 and 6 showed that the first-strand transfer (jump) step did not markedly affect the reverse transcription efficiency of HTLV-I and BLV.

A recent report by Morcock et al. (2002) suggested that the fourth possibility cannot be ruled out. Morcock et al. (2002) examined the affinity of NC proteins of retroviruses for their RNA: an NC protein of HTLV-I, p15, is more than 100-fold more loosely associated with HTLV-I RNA than NC proteins of other retroviruses, for example BLV, HIV-1 or simian immunodeficiency virus. Namely, there is a possibility that reverse transcriptase can read through UV-damaged viral RNA more readily if viral RNA is tightly associated with NC protein. Another possibility may be that RNA tightly associated with NC protein is less vulnerable to UV irradiation than that loosely associated with NC protein, like HTLV-I RNA. The D10 of BLV was still smaller than that of HIV-1, as reported by others (Yoshikura, 1989) and according to the report by Morcock et al. (2002). NC protein of BLV is still less tightly associated with viral RNA than that of HIV-1. These findings seems to favour the fourth possibility.

Lastly, the recombination of virus genomes during reverse transcription could partly explain the results obtained for BLV. Retroviruses contain two equivalent RNA genomes in their virions. It has been reported that chimeric viruses are readily made in retroviruses when cells are infected with different types of virus (Telesnitsky & Goff, 1997). If recombination between two virus genomes in the same virions takes place during reverse transcription, UV-damaged lesions in one RNA strand may be rescued by the other intact strand. Even if this is the case, HTLV-I should lack this recombination ability, since its UV sensitivity can be explained by its target genome size, as shown in Figs 4 and 6. In contrast, other retroviruses, including BLV, may have this activity.

We showed that, among the retroviruses examined so far, HTLV-I had the most UV-sensitive infectivity and that the reverse transcription of all the regions of the HTLV-I genome was similarly sensitive to UV irradiation, whereas there was a discrepancy between the UV target size of the BLV genome and its sensitivity to UV irradiation. The mechanisms underlying these observations remain to be investigated further. Our findings may be helpful to solve an old question: why are retroviruses so resistant to UV irradiation?


   ACKNOWLEDGEMENTS
 
We thank Drs G. Nolan, R. Yanagihara and D. G. Blair for kindly supplying us with Phoenix-E cells, HTLV-IMEL5-producing SI-5 cells and PG-4 cells, respectively. This work was supported in part by a Grant-in-Aid from the Japanese Society for the Promotion of Science, and grants from the Japan Health Sciences Foundation and CREST.


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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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Received 18 August 2003; accepted 30 March 2004.



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