VIROLOGY
189, 812416
(1992)
Constitutive
Synthesis L.
Dipartimento
of Polyoma 0.
O-ITAVIO,
di Biopatologia
Umana.
Antisense
L. RICCI, C.
STHANDIER, Sezione
Received
di Biologia March
RNA Renders Cells Immune PASSANANTI,’
Cellulare,
2, 1992; accepted
Universit3 May
AND di Roma
P.
to Virus Infection
AMAT?
La Sapienza,
00 16 1 Roma,
Italy
14, 1992
Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting POlyOfIIa.
0 1992 Academic
Press,
IX.
partially inhibiting the replication of adenovirus (6) and several retroviruses (6-9). The use of constitutive asRNA synthesis to render whole organisms resistant to specific viral infection has been successfully achieved in plants (10) and very recently in cells derived from transgenic rabbits expressing asRNA to adeno 5 viral early genes (11). Therefore, we decided to establish a valid animal system by previously defining an efficient in vitro cell culture viral resistance. In the present communication, we report that murine fibroblast cell lines, stably transfected with vectors expressing a specific asRNA complementary to polyomavirus (Py) early mRNAs, acquired resistance to Py infection. The cloning strategy was to insert the desired Py fragments (Fig. 1A) into pRSV-neo (12) at the 3’ of the gene coding for neomycin resistance, under the control of the Rous Sarcoma virus LTR (Fig. 1 B). The three cloned fragments (the 0.4-, 0.8-, and the 1.2-kb; see Fig. 1 B) were chosen in order to identify which sequences are the most likely to have an inhibitory effect on the expression of Py early expression by their antisense transcription. In fact the fragment 0.4 covers the ori, the CAP site, and the ATG, whereas the 0.8 fragment includes all the donor and acceptor sites for splicing of the Py early mRNA. The 1.2 fragment is the sum of those fragments. The three RSV-neo-Py AS (antisense), the RSV-neoPy 1.2 sense (S) and the RSV-neo control constructs were transfected in mouse 3T3 fibroblasts and neomycin-resistant cells were selected. Mixed cultures were tested for their susceptibility to Py infection at a multiplicity (m.0.i.) of 3 plaque-forming units (PFU). Only the plasmid coding for the 1.2-kb asRNA gave a significant
Since the first report of Zamecnik and Stephenson (1) showing the possibility of inhibiting Rous Sarcoma virus replication in cell culture using oligonucleotides complementary to viral RNA, antisense RNA and DNA have been extensively used to interfere with the expression of specific genes. Two main approaches have been followed: the use of synthetic oligonucleotides complementary to specific regions of a known mRNA, and the synthesis of antisense RNA from DNA cloned in inverted orientation (for reviews, see Refs. 1-3). The inhibition of gene expression by antisense sequences may occur at the cytoplasmic or nuclear level by different mechanisms: (i) rapid degradation of RNA-RNA or RNA-DNA duplexes, (ii) interference with mRNA processing, and (iii) inhibition of mRNA translation. A fundamet-ltal difference between the use of oligonucleotides and the synthesis of antisense RNA (asRNA) is the stability of the process. In the first case, the inhibition is generally more efficient but transient, whereas in the second it is stable over time. Both of these approaches have been used to regulate endogenous gene expression or to inhibit viral expression and replication. The approach based on the stable synthesis of asRNA seems to be very promising, possibly conferring stable molecular resistance to viral infection to both cells and organisms (4). In this case the constitutive expression of an asRNA will not interfere with the expression of endogenous genes or the normal functioning of the cell. This method has been successful in
’ On leave CNR. Roma. ’ To whom 0042.6822192 Copyright All rights
of absence reprint
from
requests
lstituto should
$5.00
Q 1992 by Academic Press. Inc. of reproduction in any form resewed.
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be addressed. 812
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FIG. 1. Scheme of the Py-cloned fragments and plasmid construction. (A) Genetrc map of Py (13) and of the cloned fragment. The 1.2-kb Pvull fragment (nt. 562 l-l 167) covers ORI, CAP, ATG, and all the splicing sites, whereas the 0.4-kb Pvull-Accl fragment (nt. 562 l-367) includes only ORI, CAP, and ATG and the 0.8-bp. Accl-Pvull fragment (nt. 368-l 167) correspond only to the splictng sites. (B) Scheme of the plasmid construction. The three polyoma fragments diagramed in Fig. 1 A (after filling of the Accl site) were cloned in both sense (S) and antisense (AS) orientations tn the HPA I site of pRSV-neo (12). pRSV-neo-Py, 1.2 S and AS; RSV-neo-Py, 0.8 S and AS; and pRSV-neo-Py. 0.4 S and AS.
resistance to Py infection determined either by Py large T antigen (PyLT) immunostaining at 2 days or by cell death at 5 days from infection (Table 1). The viral replication was measured by T antigen immunostaining and by cell death. These measurements are not subject to artifacts due to viral reabsorption or delay of viral maturation, ensuring that both early (T antigen expression) and late functions (cell death) have been exTABLE
1
SENSITIVIW OF THE MIXED TRANSFECTED CELLS WITH PLASMIDS CARRYING THE FRAGMENTS OF PY GENOME IN SENSE AND ANTISENSE ORIENTATION Cell type@ Control (RSV-neo) 1.2 s 1.2 AS 0.8 AS 0.4 AS
96 Positive
17.5 16.2 6.2 18.2 17.3
cellsb
+ 2.1 z!z 1.8 f 2.3 + 2.2 f 1.7
MortalityC
+++ +++ + +++ +++
a Mixed neomycin-resistant populations of cells transfected with the RSV-neo and different fragments of Py genome. * Percent of T antigen positive cells at 48 hr from infection. c Cell death (as measured by direct observation of cytophathic effect and by trypan bleu staining) at 5 days from infection. +++, extensive death (r95%); +, few dead cells (~20%).
pressed. The approximate correspondence of percent of T antigen positive cells to viral titer (as PFU/ml) is at 2 days1%for2X103andat5days1%for5X104and approximately 1 O* for total death. The transcription of the three antisense constructs and the control RSV-neo-Py 1.2 S was determined by Northern analysis (Fig. 2). Results show that the lack of activity of the 0.4- and 0.8-kb fragments was not due to absence of the relative asRNAs. Clones selected from either the 1.2-S or AS mixed population were analyzed for resistance to Py infection. The three 1.2-S clones showed the same sensitivity as the mixed population. The six 1.2-AS clones showed a higher level of resistance (1.4 to 3.6% of T antigen positive cells at 48 hr from Py infection). In order to determine the degree of immunity conferred by the 1.2-AS transcript, cells of one 1.2-S (S 1 -B) and one 1.2-AS (AS 2-D) clones were infected at different m.o.i. and PyLT expression was determined by immunostaining at 2 and 5 days. The S 1-B cells were fully permissive even at a m.o.i. of 1; AS 2-D cells were immune to Py infection up to a m.o.i. of 5. At a m.o.i. of 20 significant resistance was still detectable in AS 2-D cells (Table 2). To show that the observed resistance of AS clones was not due to impaired infection, we measured the viral DNA in nuclear extracts (16) at 8 to 72 hr after Py
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2-D
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FIG. 3. Southern analysis of Py DNA extracted from nuclei by Hirt’s method (16) at different intervals after infection (m.o.i. = 3). M, marker; 1 -B, clone S 1-B; and 2-D, clone AS 2-D. Numbers refer to hr after infection.
FIG. 2. Northern analysis of mixed cultures of cells transfected with different RSV-neo Py constructs. Total RNA extracted (20 pg) following the method of Chomczynski and Sacchi (14) were analyzed by Northern blotting (75) using the 1.2-kb probe. Lane 1, pRSV-neo Py 1.2 AS; lane 2, pRSV-neo Py 1, 2 S; lane 3, pRSV-neo Py 0.8 AS; lane 4, pRSV-neo Py 0.4 AS.
infection of S 1-B and AS 2-D cells. About the same amount of viral DNA was found in the nuclei of both clones at 6 hr. However, Py replicated afterward in S 1-B cells, whereas it did not replicate in AS 2-D cells (Fig. 3). Furthermore, AS 2-D cells were cotransfected
TABLE PYLT IMMUNOSTAINING
2
OF CELL CLONES AFTER Py INFECTION AT DIFFERENT m.o.i. % Positive
m.0.i. 1 3 5 20
Clones? 1-B 2-D 1-B 2-D 1-B 2-D 1-B 2-D
S AS S AS S AS S AS
2 days 5.0 +0.8 t 13.2 + 1.2 k 24.0 + 1.9 + >50 14.5 +
0.8 (+/-) 0.3 (-) 1.7 (+) 0.6 (-) 2.2 (+) 0.7 (-) (++) 2.2 (+)
cellsb 5 days >50 (++) 1 .l + 0.3 (-) dead 1.6 f 0.7 (-) dead 3.6 + 0.7 (+/-) dead >50 (++)
a Neomycin-resistent clones transfected with the RSV-neo Py 1.2 S and AS. b Percent of T antigen positive cells at 2 and 5 days from infection. In parenthesis, cell death; ++, consistent death (>50%); +, few dead cells (120%); and +/-, very few dead cells (~5%).
with pSVhygro, which contains the gene for resistance to hygromycin B (kindly provided by S. Pellegrini, Institut Pasteur, Paris, France) and pSVL Py 1.2 S or AS at a 1-to-l 0 ratio. This latter plasmid is the pSVL expression plasmid (Pharmacia) in which the Py 1.2-kb fragment in both orientations was cloned at the polylinker Smal site. After selection with hygromycin, doubly transfected cells (AS 2-D/S and AS 2-D/AS) were obtained and infected with Py at different m.o.i. The AS 2-D/S cells recovered sensitivity to viral infection (8.3 + 0.7% PyLT positive (+) cells at a m.o.i. of 3), while AS 2-D/AS cells clearly showed increased resistance to Py infection (< 0.1% PyLT + cells at a m.o.i. of 3 and 2.8 + 0.5% at a m.o.i. of 20). At 5 days after infection the difference was more evident since AS 2-D/AS did not show any PyLT+ cells at a m.o.i. of 3, whereas the AS 2-D/S showed a 20-fold increase of PyLT+ cells compared to that of AS 2-D. Therefore, the effect of asRNA was neutralized by concomitant expression of sense RNA, indicating that resistance was due to the presence of asRNA and not to impaired infection. We measured the copy number of inserts in S-l Band AS 2-D-transfected clones by digesting total cell DNA with Sacl restriction nuclease (a single cutter of the plasmid) and analyzing by Southern blot (15). Both contained single insertions (Fig. 4A). In our interpretation the upper band in lane 2 is due to a partial digestion by the restriction enzyme. A Northern analysis using the neo gene as probe was performed to measure the size of S-l B and AS 2-D transcripts and those of cells carrying the 0.8 and 0.4 fragments in AS orientation. This was done in order to compare them with that of the neo transcript of the used vector (Fig. 4B). The
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and Northern analyses of clones S 1 -B, AS 2-A, FIG. 4. Southern and AS 2-D. (A) Genomic DNA (10 pg) digested with Sac1 (single cutting of the plasmid at the insert) was analyzed by Southern blotting (15) using the 1.2.kb Py fragment as the probe, 1, S 1-B; 2, AS 2-A; and C. Control. (B) Total RNA extracted (20 pg) following the method of Chomczynski and Sacchi (14) was analyzed by Northern blotting (15) using as probe the Hindll-BarnHI neo fragment of RSVneo plasmid. 1, S 1-B; 2, AS 2-A; 3, 0.8.kb AS; 4, 0.4-kb AS: 5, RSV-neo; and M, RNA molecular weight marker (Boehringer-Mannheim, Marker I).
estimated size of the transcripts roughly corresponded to the expected size by summing the 0.4-, 0.8-, and 1.2-kb to the transcript of the neo gene. One possible mechanism for resistance to viral infection is the induction of interferon by doublestranded RNA (I 7). However, the resistance of clone AS 2-D was not due to interferon production, since no difference was observed by comparing the sensitivity to Py infection in the presence and absence of anti-interferon (D + p) antibodies. Furthermore, superinfecting encephalomyocarditis virus (18) grew equally well in Py-infected S and AS cell clones (data not shown). The partial inhibition of viral replication by stable expression of an asRNA complementary to early transcript has been successfully achieved for Adenovirus (5) and the retroviruses RSV (6, 19), HLTV-1 (9) and HIV-1 (7, 8). In other cases, such as that of influenza virus, the expression of asRNA failed to inhibit viral growth (20). The reason for the different results obtained to date could be ascribed to several factors, such as the dimension and the localization of the asRNA, its relative amount in respect to the mRNA, the stability of both asRNA and mRNA, and the maturation (splicing) of the asRNA. There is no conclusive data as to which asRNA might be the most efficient. However, the CAP 5’ untranslated, and ATG sites and the donor
and acceptor splicing sequences have been suggested as better targets for inhibition by oligonucleotides (2 1). Our present results show that the stable synthesis of an asRNA of 1.2 kb inhibits Py growth. This covers a segment of 0.4 kb that includes the ORI, CAP, and ATG sites and a segment of 0.8 kb and also the splicing sites. By expressing the 0.8- or the 0.4-kb fragments separately, we did not obtain inhibition of Py replication despite the comparable degree of transcription. This observation suggests that the ORI, CAP, and ATG sites plus the splicing sites are, in our case, all required for the inhibitory activity of Py asRNA. Total RNA extracted from transfected clones analyzed by Northern blot (Figs. 2 and 4) showed fulllength transcripts. In fact, the transcript of the three cloned fragments fused to the neomycin resistance gene are in size agreement with the expected unspliced RNA. The 1.2-kb fragment in S orientation codes for an RNA that has the donor and the receptor sites for splicing a 386-base intron (13). This observation raises the problem that the splicing signals could be strongly influenced by the surrounding context. Work is in progress to elucidate this question It has been shown that at late time of Py infection the transcription proceeds in inverse orientation to the early transcript and that it often does not stop at the polyadenylation site (22). Therefore, a similar inhibition of early transcript as we describe could be expected. However, no such effect has ever been reported. This discrepancy could well be ascribed to the stability of our transcript and/or its ability to migrate into the cytoplasm. High-level expression of asRNA is another important factor in efficient inhibition of Py replication. In fact, we have shown that a double AS/AS transfection determines a significant increase of viral resistance. Expression may be greatly increased by further development of appropriate vectors, and is therefore one of our present goals. This could make it possible to obtain in viva with transgenic animals the results observed in vitro. The resistance to a specific virus of cells transfected with vectors expressing asRNA may represent an experimental approach to protecting cells orwhole organisms at risk.
ACKNOWLEDGMENTS We are grateful to Dr. R. Baserga for supplying us with pRSV-neo. We thank Mr. L. De Angelis for technical help. Thus work was supported by P. F. Biotecnologra e Biostrumentazrone, CNR, Roma; Associazrone ltaliana Ricerca sul Cancro, Milano; and the Minister0 della Sanita-lstituto Superiore di Sanita-programma AIDS. L.O. hold a AIDS program research fellowship.
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REFERENCES 1. ZAMECNIK, P. C., and STEPHENSON, M. L., Proc. Nat/. Acad. SC;. USA 75, 280-284 (1987). 2. VAN DER KROL, A. R., MOL, 1. N. M., and STUITJE, A. R., Biofechniques 6, 958-976 (1988). 3. WEINTRAUB, H. M., Sci. Am 262, 34-40 (1990). 4. BALTIMORE, D., Nature (London) 335, 395-396 (1988). 5. MIROSHNICHENKO, 0. I., PONOMAREVA. T. I., and TIKCHONENKO, T. I., Gene 84, 83-89 (1989). 6. CHANG, L. J., and STOLLFUS. C. M., /. Viral. 61, 92 l-924 (1987). 7. RHODES, A., and JAMES, W., /. Gen. Viral. 71, 1965-l 974 (1990). 8. SCZAKIEL, G., and PAWLITA, M.. /. Viral. 65, 468-472 (1991). 9. VON RUDEN, T., and GILBOA, E., /. Viral. 63, 677-682 (1989). 10. HEMENWAY, C., FANG, R. X., KANIESWSKI. W. K., CHUA, N. H., and TUMER, N. E., fMBO/. 7, 1273-l 280 (1988). 11. ERNST, L. K., ZAKEHARENKO, V. I., SURAEVA, N. M., PONOMAREVA, T. I., MIROSHNICHENKO, 0. I., PROKOF’EV, M. I., and TIKCHONENKO, T. I., Theriogenology35, 1257-1271 (1991). 12. MULLIGAN, R., and BERG, P., Science 209, 1422-1427 (1980). 13. TOOZE. 1. (ed.), Molecular Biology of Tumor Viruses: DNA Tumor
14. 15.
16. 17. 18. 19. 20. 21.
22.
Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981. CHOMCZYNSKI, P., and SACCHI, N., Anal. Biochem. 162,156-l 59 (1987). SAMBROOK, J., FRITSCH, E. F., and MANIATIS, T., Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989. HIRT, B., /. n/lo/. Biol. 26, 365-369 (1967). LENGYEL, P., Annu. Rev. Biochem. 51, 25 l-282 (1982). BAGLIONI, C., and NILSEN, T. W., ln “Interferon” (I. Gesser, Ed.), Vol. 5, pp. 23-42. Academic Press, London, New York, 1984. To, R. Y., BOOTH, S. C., and NEIMAN, P., &lo/. Cell. Bio. 6,47584762 (1986). LEITER, 1. M. E., KRYSTAL, M., and PALESE, P., Virus Research 14, 141-160 (1989). GOODCHILD, J. S., AGRAWAL, S., CIVEIRA, M. P., SARIN, P. S., SUN, D., and ZAMECNIK, P. C., Proc. Nat/. Acad. Sci. USA 85, 5507551 1 (1988). ACHESON, N. H., Proc. Natl. Acad. Sci. USA 75, 4754-4758 (1978).
189, 812416
(1992)
Constitutive
Synthesis L.
Dipartimento
of Polyoma 0.
O-ITAVIO,
di Biopatologia
Umana.
Antisense
L. RICCI, C.
STHANDIER, Sezione
Received
di Biologia March
RNA Renders Cells Immune PASSANANTI,’
Cellulare,
2, 1992; accepted
Universit3 May
AND di Roma
P.
to Virus Infection
AMAT?
La Sapienza,
00 16 1 Roma,
Italy
14, 1992
Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting POlyOfIIa.
0 1992 Academic
Press,
IX.
partially inhibiting the replication of adenovirus (6) and several retroviruses (6-9). The use of constitutive asRNA synthesis to render whole organisms resistant to specific viral infection has been successfully achieved in plants (10) and very recently in cells derived from transgenic rabbits expressing asRNA to adeno 5 viral early genes (11). Therefore, we decided to establish a valid animal system by previously defining an efficient in vitro cell culture viral resistance. In the present communication, we report that murine fibroblast cell lines, stably transfected with vectors expressing a specific asRNA complementary to polyomavirus (Py) early mRNAs, acquired resistance to Py infection. The cloning strategy was to insert the desired Py fragments (Fig. 1A) into pRSV-neo (12) at the 3’ of the gene coding for neomycin resistance, under the control of the Rous Sarcoma virus LTR (Fig. 1 B). The three cloned fragments (the 0.4-, 0.8-, and the 1.2-kb; see Fig. 1 B) were chosen in order to identify which sequences are the most likely to have an inhibitory effect on the expression of Py early expression by their antisense transcription. In fact the fragment 0.4 covers the ori, the CAP site, and the ATG, whereas the 0.8 fragment includes all the donor and acceptor sites for splicing of the Py early mRNA. The 1.2 fragment is the sum of those fragments. The three RSV-neo-Py AS (antisense), the RSV-neoPy 1.2 sense (S) and the RSV-neo control constructs were transfected in mouse 3T3 fibroblasts and neomycin-resistant cells were selected. Mixed cultures were tested for their susceptibility to Py infection at a multiplicity (m.0.i.) of 3 plaque-forming units (PFU). Only the plasmid coding for the 1.2-kb asRNA gave a significant
Since the first report of Zamecnik and Stephenson (1) showing the possibility of inhibiting Rous Sarcoma virus replication in cell culture using oligonucleotides complementary to viral RNA, antisense RNA and DNA have been extensively used to interfere with the expression of specific genes. Two main approaches have been followed: the use of synthetic oligonucleotides complementary to specific regions of a known mRNA, and the synthesis of antisense RNA from DNA cloned in inverted orientation (for reviews, see Refs. 1-3). The inhibition of gene expression by antisense sequences may occur at the cytoplasmic or nuclear level by different mechanisms: (i) rapid degradation of RNA-RNA or RNA-DNA duplexes, (ii) interference with mRNA processing, and (iii) inhibition of mRNA translation. A fundamet-ltal difference between the use of oligonucleotides and the synthesis of antisense RNA (asRNA) is the stability of the process. In the first case, the inhibition is generally more efficient but transient, whereas in the second it is stable over time. Both of these approaches have been used to regulate endogenous gene expression or to inhibit viral expression and replication. The approach based on the stable synthesis of asRNA seems to be very promising, possibly conferring stable molecular resistance to viral infection to both cells and organisms (4). In this case the constitutive expression of an asRNA will not interfere with the expression of endogenous genes or the normal functioning of the cell. This method has been successful in
’ On leave CNR. Roma. ’ To whom 0042.6822192 Copyright All rights
of absence reprint
from
requests
lstituto should
$5.00
Q 1992 by Academic Press. Inc. of reproduction in any form resewed.
di Tecnologie
Biomediche,
be addressed. 812
SHORT
COMMUNICATIONS
813
1.2 Kb
B pq
FIG. 1. Scheme of the Py-cloned fragments and plasmid construction. (A) Genetrc map of Py (13) and of the cloned fragment. The 1.2-kb Pvull fragment (nt. 562 l-l 167) covers ORI, CAP, ATG, and all the splicing sites, whereas the 0.4-kb Pvull-Accl fragment (nt. 562 l-367) includes only ORI, CAP, and ATG and the 0.8-bp. Accl-Pvull fragment (nt. 368-l 167) correspond only to the splictng sites. (B) Scheme of the plasmid construction. The three polyoma fragments diagramed in Fig. 1 A (after filling of the Accl site) were cloned in both sense (S) and antisense (AS) orientations tn the HPA I site of pRSV-neo (12). pRSV-neo-Py, 1.2 S and AS; RSV-neo-Py, 0.8 S and AS; and pRSV-neo-Py. 0.4 S and AS.
resistance to Py infection determined either by Py large T antigen (PyLT) immunostaining at 2 days or by cell death at 5 days from infection (Table 1). The viral replication was measured by T antigen immunostaining and by cell death. These measurements are not subject to artifacts due to viral reabsorption or delay of viral maturation, ensuring that both early (T antigen expression) and late functions (cell death) have been exTABLE
1
SENSITIVIW OF THE MIXED TRANSFECTED CELLS WITH PLASMIDS CARRYING THE FRAGMENTS OF PY GENOME IN SENSE AND ANTISENSE ORIENTATION Cell type@ Control (RSV-neo) 1.2 s 1.2 AS 0.8 AS 0.4 AS
96 Positive
17.5 16.2 6.2 18.2 17.3
cellsb
+ 2.1 z!z 1.8 f 2.3 + 2.2 f 1.7
MortalityC
+++ +++ + +++ +++
a Mixed neomycin-resistant populations of cells transfected with the RSV-neo and different fragments of Py genome. * Percent of T antigen positive cells at 48 hr from infection. c Cell death (as measured by direct observation of cytophathic effect and by trypan bleu staining) at 5 days from infection. +++, extensive death (r95%); +, few dead cells (~20%).
pressed. The approximate correspondence of percent of T antigen positive cells to viral titer (as PFU/ml) is at 2 days1%for2X103andat5days1%for5X104and approximately 1 O* for total death. The transcription of the three antisense constructs and the control RSV-neo-Py 1.2 S was determined by Northern analysis (Fig. 2). Results show that the lack of activity of the 0.4- and 0.8-kb fragments was not due to absence of the relative asRNAs. Clones selected from either the 1.2-S or AS mixed population were analyzed for resistance to Py infection. The three 1.2-S clones showed the same sensitivity as the mixed population. The six 1.2-AS clones showed a higher level of resistance (1.4 to 3.6% of T antigen positive cells at 48 hr from Py infection). In order to determine the degree of immunity conferred by the 1.2-AS transcript, cells of one 1.2-S (S 1 -B) and one 1.2-AS (AS 2-D) clones were infected at different m.o.i. and PyLT expression was determined by immunostaining at 2 and 5 days. The S 1-B cells were fully permissive even at a m.o.i. of 1; AS 2-D cells were immune to Py infection up to a m.o.i. of 5. At a m.o.i. of 20 significant resistance was still detectable in AS 2-D cells (Table 2). To show that the observed resistance of AS clones was not due to impaired infection, we measured the viral DNA in nuclear extracts (16) at 8 to 72 hr after Py
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FIG. 3. Southern analysis of Py DNA extracted from nuclei by Hirt’s method (16) at different intervals after infection (m.o.i. = 3). M, marker; 1 -B, clone S 1-B; and 2-D, clone AS 2-D. Numbers refer to hr after infection.
FIG. 2. Northern analysis of mixed cultures of cells transfected with different RSV-neo Py constructs. Total RNA extracted (20 pg) following the method of Chomczynski and Sacchi (14) were analyzed by Northern blotting (75) using the 1.2-kb probe. Lane 1, pRSV-neo Py 1.2 AS; lane 2, pRSV-neo Py 1, 2 S; lane 3, pRSV-neo Py 0.8 AS; lane 4, pRSV-neo Py 0.4 AS.
infection of S 1-B and AS 2-D cells. About the same amount of viral DNA was found in the nuclei of both clones at 6 hr. However, Py replicated afterward in S 1-B cells, whereas it did not replicate in AS 2-D cells (Fig. 3). Furthermore, AS 2-D cells were cotransfected
TABLE PYLT IMMUNOSTAINING
2
OF CELL CLONES AFTER Py INFECTION AT DIFFERENT m.o.i. % Positive
m.0.i. 1 3 5 20
Clones? 1-B 2-D 1-B 2-D 1-B 2-D 1-B 2-D
S AS S AS S AS S AS
2 days 5.0 +0.8 t 13.2 + 1.2 k 24.0 + 1.9 + >50 14.5 +
0.8 (+/-) 0.3 (-) 1.7 (+) 0.6 (-) 2.2 (+) 0.7 (-) (++) 2.2 (+)
cellsb 5 days >50 (++) 1 .l + 0.3 (-) dead 1.6 f 0.7 (-) dead 3.6 + 0.7 (+/-) dead >50 (++)
a Neomycin-resistent clones transfected with the RSV-neo Py 1.2 S and AS. b Percent of T antigen positive cells at 2 and 5 days from infection. In parenthesis, cell death; ++, consistent death (>50%); +, few dead cells (120%); and +/-, very few dead cells (~5%).
with pSVhygro, which contains the gene for resistance to hygromycin B (kindly provided by S. Pellegrini, Institut Pasteur, Paris, France) and pSVL Py 1.2 S or AS at a 1-to-l 0 ratio. This latter plasmid is the pSVL expression plasmid (Pharmacia) in which the Py 1.2-kb fragment in both orientations was cloned at the polylinker Smal site. After selection with hygromycin, doubly transfected cells (AS 2-D/S and AS 2-D/AS) were obtained and infected with Py at different m.o.i. The AS 2-D/S cells recovered sensitivity to viral infection (8.3 + 0.7% PyLT positive (+) cells at a m.o.i. of 3), while AS 2-D/AS cells clearly showed increased resistance to Py infection (< 0.1% PyLT + cells at a m.o.i. of 3 and 2.8 + 0.5% at a m.o.i. of 20). At 5 days after infection the difference was more evident since AS 2-D/AS did not show any PyLT+ cells at a m.o.i. of 3, whereas the AS 2-D/S showed a 20-fold increase of PyLT+ cells compared to that of AS 2-D. Therefore, the effect of asRNA was neutralized by concomitant expression of sense RNA, indicating that resistance was due to the presence of asRNA and not to impaired infection. We measured the copy number of inserts in S-l Band AS 2-D-transfected clones by digesting total cell DNA with Sacl restriction nuclease (a single cutter of the plasmid) and analyzing by Southern blot (15). Both contained single insertions (Fig. 4A). In our interpretation the upper band in lane 2 is due to a partial digestion by the restriction enzyme. A Northern analysis using the neo gene as probe was performed to measure the size of S-l B and AS 2-D transcripts and those of cells carrying the 0.8 and 0.4 fragments in AS orientation. This was done in order to compare them with that of the neo transcript of the used vector (Fig. 4B). The
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7,4 -
5.3 28 -
2.8 18 1,81.6.
and Northern analyses of clones S 1 -B, AS 2-A, FIG. 4. Southern and AS 2-D. (A) Genomic DNA (10 pg) digested with Sac1 (single cutting of the plasmid at the insert) was analyzed by Southern blotting (15) using the 1.2.kb Py fragment as the probe, 1, S 1-B; 2, AS 2-A; and C. Control. (B) Total RNA extracted (20 pg) following the method of Chomczynski and Sacchi (14) was analyzed by Northern blotting (15) using as probe the Hindll-BarnHI neo fragment of RSVneo plasmid. 1, S 1-B; 2, AS 2-A; 3, 0.8.kb AS; 4, 0.4-kb AS: 5, RSV-neo; and M, RNA molecular weight marker (Boehringer-Mannheim, Marker I).
estimated size of the transcripts roughly corresponded to the expected size by summing the 0.4-, 0.8-, and 1.2-kb to the transcript of the neo gene. One possible mechanism for resistance to viral infection is the induction of interferon by doublestranded RNA (I 7). However, the resistance of clone AS 2-D was not due to interferon production, since no difference was observed by comparing the sensitivity to Py infection in the presence and absence of anti-interferon (D + p) antibodies. Furthermore, superinfecting encephalomyocarditis virus (18) grew equally well in Py-infected S and AS cell clones (data not shown). The partial inhibition of viral replication by stable expression of an asRNA complementary to early transcript has been successfully achieved for Adenovirus (5) and the retroviruses RSV (6, 19), HLTV-1 (9) and HIV-1 (7, 8). In other cases, such as that of influenza virus, the expression of asRNA failed to inhibit viral growth (20). The reason for the different results obtained to date could be ascribed to several factors, such as the dimension and the localization of the asRNA, its relative amount in respect to the mRNA, the stability of both asRNA and mRNA, and the maturation (splicing) of the asRNA. There is no conclusive data as to which asRNA might be the most efficient. However, the CAP 5’ untranslated, and ATG sites and the donor
and acceptor splicing sequences have been suggested as better targets for inhibition by oligonucleotides (2 1). Our present results show that the stable synthesis of an asRNA of 1.2 kb inhibits Py growth. This covers a segment of 0.4 kb that includes the ORI, CAP, and ATG sites and a segment of 0.8 kb and also the splicing sites. By expressing the 0.8- or the 0.4-kb fragments separately, we did not obtain inhibition of Py replication despite the comparable degree of transcription. This observation suggests that the ORI, CAP, and ATG sites plus the splicing sites are, in our case, all required for the inhibitory activity of Py asRNA. Total RNA extracted from transfected clones analyzed by Northern blot (Figs. 2 and 4) showed fulllength transcripts. In fact, the transcript of the three cloned fragments fused to the neomycin resistance gene are in size agreement with the expected unspliced RNA. The 1.2-kb fragment in S orientation codes for an RNA that has the donor and the receptor sites for splicing a 386-base intron (13). This observation raises the problem that the splicing signals could be strongly influenced by the surrounding context. Work is in progress to elucidate this question It has been shown that at late time of Py infection the transcription proceeds in inverse orientation to the early transcript and that it often does not stop at the polyadenylation site (22). Therefore, a similar inhibition of early transcript as we describe could be expected. However, no such effect has ever been reported. This discrepancy could well be ascribed to the stability of our transcript and/or its ability to migrate into the cytoplasm. High-level expression of asRNA is another important factor in efficient inhibition of Py replication. In fact, we have shown that a double AS/AS transfection determines a significant increase of viral resistance. Expression may be greatly increased by further development of appropriate vectors, and is therefore one of our present goals. This could make it possible to obtain in viva with transgenic animals the results observed in vitro. The resistance to a specific virus of cells transfected with vectors expressing asRNA may represent an experimental approach to protecting cells orwhole organisms at risk.
ACKNOWLEDGMENTS We are grateful to Dr. R. Baserga for supplying us with pRSV-neo. We thank Mr. L. De Angelis for technical help. Thus work was supported by P. F. Biotecnologra e Biostrumentazrone, CNR, Roma; Associazrone ltaliana Ricerca sul Cancro, Milano; and the Minister0 della Sanita-lstituto Superiore di Sanita-programma AIDS. L.O. hold a AIDS program research fellowship.
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