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5′ avian leukosis virus sequences and osteopetrotic potential
VIROLOGY
190, 866-87
1 (1992)
5’ Avian Leukosis
H. L.
Virus Sequences
and Osteopetrotic
Potential
R. G. FosrER,t B. P. BLAIS,* S. S. REINSCH,+ M. NEWSTEIN,+ AND P. R. SHANK+
ROBINSON,*!’
Departments of *Pathology and tMolecu/ar Genetics and Microbiology, University Worcester, Massachusetts 0 1655; and +Division of Biology and Medicine, Received
April
13, 1992; accepted
of Massachusetts Brown University, June 29,
Medical Center, 55 Lake Avenue North, Providence, Rhode Island 029 12
1992
Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5’ viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5 sequences that affected osteopetrotic potential resided in an 834-bp region near the 5’ LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA. o 1992 Academic PWSS, I~C.
Avian leukosis virus (ALV) induced osteopetrosis results from the abnormal growth and differentiation of infected osteoblasts (20). The induction of osteopetrosis requires the establishment of a persistent viremia and is associated with the persistent synthesis of viral DNA in bone (3, 16). The persistent synthesis of viral DNA results in the accumulation of 2-20 copies of unintegrated plus integrated viral DNA molecules per bone cell (16, 17). The amount of accumulated viral DNA correlates with both the rapidity and the severity of osteopetrosis induction (16). Several regions of the viral genome determine osteopetrotic potential. The induction of osteopetrosis requires long terminal repeat (LTR) sequences with efficient transcriptional control elements such as those found in the LTRs of exogenousviruses. LTRs of exogenous origin differ in the efficiency with which they induce osteopetrosis. In one series of recombinants, the LTR from Rous-associated virus type 1 was more efficient than the LTR from Prague Rous sarcoma virus-B in causing osteopetrosis (10, 19). In a second series, the LTR of myeloblastosis-associated virus-2(O) had higher osteopetrotic potential than the LTR of UR2-associated virus (4). The LTR of the endogenous Rousassociated virus-O (RAV-0) is substantially less efficient than those of exogenous viruses (9). This LTR cannot support the induction of osteopetrosis (19). At least two regions of the ALV genome outside of the LTR affect osteopetrotic potential. One of these encodes envelope glycoproteins. Envelope glycoproteins that use the subgroup B/E receptor confer higher osteopetrotic potentials than envelope glycoproteins that use the subgroup A receptor (4,.6). This difference presumably reflects differences in the tissue-specific \ ’ To whom 0042.6822/92
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Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
display or activity of subgroup A and B/E receptors (7). A second region that affects osteopetrotic potential maps to a 1.4-kb region near the 5’ LTR (17). The current study was undertaken to further define the role of 5’ sequences in osteopetrotic potential. To identify 5’sequences that affect osteopetrotic potential, RAV-0 sequences were substituted for homologous 5’ sequences in Br21, a subgroup E virus with high osteopetrotic potential (19). Two independent clones of each of two constructs were constructed using conserved Sac1 and Ball sites to substitute RAV-0 fragments for Br21 fragments (Fig. 1). Plasmids designated pBrl6Os contained RAV-0 sequences from positions 255 to 1089, while pBrl5Os contained RAV-0 sequences from positions 1089 to 1630 of viral RNA (18). The presence of RAV-0 or Br21 fragments in constructs was verified using diagnostic Pstl sites (Fig. 1). gag
__
env
PO1
w LTR
LTR
MA p10 c*
NCPR
c”’
RAV-0
1
I 0
i
I 1
7
I 2
T
i
I 3
i
I 4
I 5
I 6
I 7
kb
FIG. 1. Genomes of RAW0 and Br2 1. LTR. gag, PO/, and env indicate the long terminal repeat regions and sequences encoding the Gag, reverse transcriptase, and envelope proteins respectively. Only the restriction endonuclease sites used for construction and verification of recombinants are indicated. B, Ball; P, Pstl; S, Sacl; X, Xbal. Sites used for verification are indicated with asterisks.
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OSTEOPETROSIS
LOWER LIMB PARALYSIS
GENOME ROOSTER
RAW0
HEN
SEVERE
MILD
SEVERE
0122
o/22
0132
O/32
0122
0132
2l7
2i7
NA
NA
Br21
NA
o/10
Brl3Os
Brl6Os
,gr Is-i LTRc
gag
x
11116
l/15
pal x
env
MILD
NA
o/10
6114
a
NA
NA
NA
NA
NA
0116
NA
5112
2112
NA
NA
5116
6i7
O/7
12l16
0il5
0112
l/12
2l15
Brl40s
BrlliOs
HEN
ROOSTER
O/7
b
l/l2
c
>LTR
FIG. 2. Disease potential of the parent and recombinant viruses. For construction of the Brl3Os and Brl4Os, see Ref (7 7). The Brl6Os and Brl5Os were constructed by substituting the indicated pBr21 fragments into pBrl30 (see text and Fig. 1). Two independently generated DNA clones were used for tests of the Brl30, 140, 150, and 160 recombinants. Tests conducted at different times are tabulated separately. Cases of moderate and severe osteopetrosis are grouped under severe. (a) Four of these six cases of paralysis occurred in birds with severe osteopetrosis. (b) Neither case scored for osteopetrosis. (c) Occurred in the hen with osteopetrosis. NA, not available. Sites for restriction endonucleases used in constructions: B, Ball; P, WI; S, Sacl; X, Xbal.
Plasmid DNAs were transfected into chick embryo fibroblasts (from chicken line 158 evl) for recovery of virus. Virus stocks, designated the Brl5Os and Brl6O.s contained from 1 X 1 O”-1 X 10’ fibroblast-infectious units per milliliter. In viva tests demonstrated that the Brl5Os caused much more rapid and severe osteopetrosis than the Brl6Os (Fig. 2). Tests were initiated by inoculating dayold K28 chicks with 0.2 ml of undiluted virus stocks. At 1 month postinoculation, chicks were bled and tested for viremia using an assay for particulate reverse transcriptase (15). The two test groups exhibited comparable levels of viremia (Fig. 3). By 1 month, Brl50s-inoculated chickens had begun to show signs of osteopetrosis. By 2 months, 74% of the Brl5Os but only 4% of the Brl60s-inoculated birds had developed severe osteopetrosis. Roosters are more susceptible to osteopetrosis than hens (16). In the Brl5Os group, all of the viremic roosters developed moderate to severe osteopetrosis. By contrast, in Brl60s-infected birds, only the roosters with the highest viremias developed moderate to severe disease (Fig. 3).
Brl50s
Brl60s
FIG. 3. Levels of viremia at 1 month postinoculation in Brl50 and Brl60inoculated chicks. Each symbol represents data for one rooster. A, roosters that developed osteopetrosis and paralysis. 0, roosters that developed only osteopetrosis. & rooster that developed only paralysis. 0, roosters that developed mild osteopetrosis or no disease.
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. Sac1 . . GAGCTCcAcgGCcgGGggccaagatacCCTaCCGAGaACTCAGAgaGTCGTtGG~GAC~g~~~GCCCgACGACTGAGCaGTCCACccCA~cgtG B~~~:------T-CT--*G--*GCCCAGATAC---A---A-----A-------GA-----T---------GA--GA-----G----------A------~~---G~G*-------GA-----T---------GA--GA-----G----------A------CC---G~cRAV-O:------C-CD--CG--GCCCMGATAA---A----A-----A ev-2 : ev-1: P~C:------T-CT--AG--AGCCCAGATAC---A-----A-------GA-----T---------GA--GA-----G----------A------CC---GCGTMC29:------C-CG--CG--GGGCAAGATAA---A---A-----A-------GA-----T---------GA--GA-----G----------A------CC---GCGTFSV:------C-CG--CG--GGCC~GAT~---------A----------A------CC---GCGTAEV-R:------T-CT--AG--GGCCAAGATAC---A---A-----A-------TA-----T---------GA--GA-----G----------A------AC---GTGCSRA:------C-GG--CC--AGCGACTGACC---G-----A-------GG-----~---------AG--TG-----G----.........----CC---G~A~y73:------C-CG--CG--GGCCAAGATAA---A---A-----A-------GA-----T---------GA--GA-----G----------A------CT---G*C---A-----C-------GA-----T---------GA--GA-----G----------G------CC---ACGTUR2:------T-CT--AG--AGCCAACATAC
*
v
4-
.
atTctGGTCgcCCgG Br21:AT-CT----GC--GWV-O:AT-CC----GC--Tev-2 : ev-1: PrC:AT-CT----GC--GMC29:AT-CT----GC--GFSV:AT-CT----GC--GAEV-R:AT-CC----AC--.SRA:TC-TT----GG--TY73:AT-CC----GC--TURZ:AT-CT----GC--G-
--------------------------------C--G----C---------------------A--------------------------------C--G----C------------------------G-------A--------------------------------
C--G----C-----------------------------------------------C--G----C----------------------------------------------C--A----C----------------------------------------------C--G----C-------------------------------------------G--G----C-------------------------------------------C--G----T-------------------
*
*
. ATaGGGGCCATGtTGTCCCtctTACAAAAgGAAGGGTTGcTTATGTctccCTCAGAcTTATATTCcCCGGGGTCcT~ATCCCAT~CcGCGGCg~ct .
.
Br21:--A---------T------TCT-------G ~V-O:--A---------T-
.
.
.
.
-----------------TCC------C------C---C--------C----
.
554
.
654
-------T--C-----G--A
-----TGT-------G-----------------TCC------C--------C--------C-----------T--C-----G--*
--------T--C-----G--CT
ev-2 : ev-l:--A---------T------TGT-------G -----------------TCC------C------C---C--------C-----------T--C-----G--~ PrC:--A---------T------TCT----G----G----------------TCC------C------C---C--CMC29:--A---------T------TCT----G -----------------Tcc------c------~---c--------A-----------T--c-----A--cA FSV:--A---------T------AGT----G----G-----------------CCT------C--------C--------C-----------T--C-----A--CA AEV-R:--A---------C------TGC----G----G-----------------TCC------C--------C--------C-----------T--C-----G--~ SRA:--A---------T------TGT-----G---G-----------------TCC------T--------T--------C-----------C--T-----G--~ Y73:--A---------C--
----TGT-------G-----------------TCC------C--------C------C---C-----------T--C-----G--~
UR2:--C---------T------TCT-------A-----------------TCC------C--------C---
.
x- *
.
.
.
.
-----C-----------C--T---G--CT
.
*
.
CcCAGCGGGCaATGgTACTTGGgAAATCGGGGAGAGTT~C~GGGGATTGG~TTGGgGGCA~~gGC~CtCGAGA~~CA~~ACAT~GA c--------T---A-------G-------------------------------------G-------A-G-----T-----------------------
Br21::-C--------A---G-------G------------------------------------G-------C-G-----T----------------------RAV-0 _-~ -------------------------------G-------A-G-----T----------------------ev-2 :-C--------A---G-------G-----C--------A---G-------G-------------------------------------G-------A-G-----T----------------------ev-l:PrC :-C--------T---A-------G-------------------------------------G-------A-G-----T----------------------------------------------------------G-------A-G-----T----------------------MC29 :-C--------A---G-------GFsV:-A--------G---G-------G -------------------------------------G-------A-G-----C---------------------------T---G-------G------------------------------------AEV-R:-C--SRA:-C--------A---G------A-------------------------------------G-------A-G-----T----------------------Y73 :-C--------A---G-------G------------------------------------uR2 :-C--------A---G-------G-------------------------------------
XhOI
C-------A-G-----T----------------------G-------A-G-----T----------------------G-------A-A-----C-----------------------
FIG. 4. Sequence data for the Sacl-to-Ball fragments of RAV-0 and Br21 compared with those for other avian leukosis sarcoma viruses. Numbers refer to positions relative to the sequence of Prague Rous sarcoma virus C RNA (18). A consensus sequence is presented above the comparisons. Uppercase letters in the consensus and dashes in viral sequences indicate bases present in all sequenced isolates. Differences between RAV-0 and Br21 are highlighted by asterisks, The positions of restriction endonuclease sites, the splice donor (XD) for subgenomic viral RNAs, and boundaries for MA, ~10, and CA are indicated above the consensus. TSR, a highly conserved translational start region is boxed. The gag enhancer and the NRS regulatory sequences for splicing are bounded by arrows, as indicated. 7 indicates insertions into the sequence of one nucleotide (T) at position 353 in MC29 and of six nucleotides (ACAGCC) at position 883 in SRA and Y73. Data for Prague-C Rous sarcoma virus (PRC), Schmidt-Ruppin-A Rous sarcoma virus (SRA), Avian erythroblastosis virus strain-R (AEV-R), Fujinami sarcoma virus (FSV), and avian sarcoma virus Y73 were obtained from Genbank. Data for myeloblastosis virus MC29 from (74); for UR2, (73), and for ev-1 and ev-2, (22).
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r
. . . GC~GCAAAGTTTTGGTTGGGATTAGGGGGAGGGAGGGTCG sr~l:---------------------------------------------------------G-------RAv-o:--------------------------------------------------------G-ev-2:--------------------------------------------------------e"-l:--------------------------------------------------------G-----------------A--------------------A-------------------------G-----prc:---------------------------------nc29:--------------------------------------------------------FS":---------------------------------------------------------G-------------AE"-R:--------------------------------------------------------SRA:---------------------------------------------------------G-----------------T--------------------A--~,~:---------------------------------------------------------G-----------------A--------------------G--UR~:---------------------------------------------------------A-----------------A--------------------A---
NRS .
.
754
---------A--------------------A-----------------A-------------------A--G-----------------A-------------------A-------------A-------------------A--G-----------------A--------------------G-----A-------------------G---------.--------A--------------------G~~~
r _
ENHANCER * . . . . -<1 GAGGAagTGGGAGAaACAACTGtGCAGCGAGATgCG~GATGGCGCCgGAGg~acgGCCACACCT~CcG~GGCACATcCTGcTATcAtTGCGG~ S~Z~:-----AG-------A------T----------G-------------G---G--ACG--------------G-------------C-----T----------T----------G-------------G---G--ACG-------------C--------------C---T---T------RAV-O:-----AG-------A--e"-2:-----AG-------A-------T---------G-------------G---G--ACG--------------C--------------C-----T------e"-l:-----AG-------A------C----------G-------------G---A--ATG-------------C--------------C-----G------------T----------G-------------G---G--ACG--------------C--------------C-----T------~~C:-----AG-------AM~~~:-----AG-------A------T----------G-------------G---G--ACT--------------C--------------C-----T-------------A-------------G---G------------C--------------C-----T------. ..------A-------T--FSV:..... AE"-R:-----AA-------G-------T--------G---G------------G---A--ATG-------------C--------------T-----G------C--------------C-----T------SRA:-----GG-------A-------T----------G-------------A---G--GCG-------------Y,3;-----AG-------A------T----------G------------A---G--ACG-------------C--------------C---T---T------"R2:-----AG-------A-------T----------G-------------G---G--ACG --------------C----
. v . . . . r'Pl0 CAGCtatTGGCTGtAAtTGCGcCACaGCCTCgGCcCCTCCTCCtCCtTAtGTGGGGAGT~tTTGTaTCCTTCCCTGGCGGGGGtGGGaGAGCAGCaGgg
854
.
954
.
1054
NRS
ENHANCER
71.
. . . CCaGGGgGgTgAcACacCTcgGGGGGCGGAaCAGcCAAgGGcGGAGCCAgGGCaCgCGGGTCtGgCcCcTGGGCCGGCCCTGACTGACTGGGC~GGaTc
.
.
*
plo<--l-->CA . . aGGGAGGAGCTTGCGAGTACaGGTCCGCCCgTGGTqGCCATGCCTGTAGTGATTAAqACAGAGGGACCCGCcTGGACCCCTCTGGAGCCAaAattgATC B~~~:A-------------------A-------G---G----G--------------------G--------------C------------------A-ATTG--RAV-O:A-------------------A---------G----G--------------------G--------------C------------------A-ATTG--e"-2:A-------------------A---------G----G-----e"-l:A-------------------T---------G----G-----PrC:A-------------------T---------G----G--------------------G--------------T------------------A-ATTG--nC2g:G-------------------T---------G----G--------------------C--------------C------------------A-ATTG--FSV:A-------------------A-------A---A----G--------------------~--------------C------------------A-ATTG--AEV-R:A-------------------T---------G----A--------------------G--------------C------------------G-GGAC--FIG. 4-Continued
.
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The lower incidence and milder form of Brl60s-induced osteopetrosis differed in its appearance from Brl50s-induced osteopetrosis. Brl5Os caused a generalized increase in the diameter of the leg bones that is characteristic of ALV-induced osteopetrosis. By contrast, Brl6Os induced a lumpy form of osteopetrosis that was manifested as discrete foci of abnormal growth. Brl60s-induced lumps projected from the bone by a few millimeters and had widths of up to 1 cm. This form of disease had been previously noted in Brl30sinfected chickens. The Brl50s-inoculated chicks also developed a high incidence of lower limb paralysis. The appearance of this disability had been previously noted in Br21- and Brl40s-infected chicks (Fig. 2). The lower limb paralysis did not necessarily correlate with the appearance or severity of osteopetrosis (Figs. 2 and 3). Chickens with paralysis did not have joint or hip problems. Serial sections of the brain and the spinal cord of one disabled chicken did not reveal lesions. Thus, the cause-of this disability is not understood. A subgroup A recombinant that induces an intermediate incidence of osteopetrosis (pEU) causes a walking disability associated with ataxia (4). In contrast to the Brl50s-induced paralysis, the ataxia is associated with lesions in the cerebellum (4). pBr21 and pRAV-0 were partially sequenced to identify differences in the fragment that affected osteopetrotic potential. These sequence data are compared with that for other avian leukosis sarcoma viruses in Fig. 4. The 17 bases that differed between RAV-0 and Br21 are indicated with asterisks. Eleven differences were found in noncoding leader sequences. These differences were localized in poorly conserved regions of the leader immediately 3’ to the Sac1 site and immediately 5’ to the highly conserved translational start region (TSR) (5). Positions 271-275 contained changes in three of five bases that were unique to RAV-0. Other differences in the leader represented sequences present in other exogenous viruses. Six differences between RAV-0 and Br21 were present in MA codons. Four of these had no effect on amino acid usage. A codon change at position 569 resulted in a conservative change that is also present in the MA proteins of several exogenous viruses. A codon change at position 623 gave a nonconservative change (a glutamine to a lysine) that is unique to RAV-0. Studies using recombinants between Rous-associated virus-l and RAV-0 have shown that a 375-bp Sacl-to-Xhol fragment from RAV-1 confers osteopetrotic potential (6). Interestingly, this 375-bp region contains all of the unique differences in leader sequences as well as the one nonconservative amino acid difference between RAV-0 and Br21.
Functions encoded in 5’ sequences have not been correlated with osteopetrotic potential. The RAV-0 leader sequence is not deficient for gene expression (9). The leader sequences and MA proteins of osteopetrosis-inducing and non-osteopetrosis-inducing viruses do not affect the efficiency of the formation of infectious particles (unpublished observations). A gag enhancer activity (1, 8) does not distinguish osteopetrosis from non-osteopetrosis-associated sequences (I, unpublished observations). Tests for this enhancer activity have been conducted in a number of cell types including cultured osteoblasts (unpublished observations). The activity of a negative regulator of splicing (2, 12) also does not distinguish osteopetrosis and nonosteopetrosis-inducing infections in fibroblasts and osteoblasts (unpublished observations). In closing, we speculate that the osteopetrosis-enhancing function in 5’ Br21 sequences encodes a function of exogenous but not endogenous viruses. Such is consistent with our identifying osteopetrosis-potentiating functions in 5’ sequences in two series of recombinants between exogenous and endogenous viruses (Fig. 2, Ref. 6). Such is also consistent with recombinants between exogenous viruses failing to reveal a role for 5’ internal sequences in osteopetrosis induction (4). Thus, the 5’ sequences that affect osteopetrotic potential may represent yet another example of a function that has evolved differently in endogenous and exogenous viruses.
ACKNOWLEDGMENTS We are indebted to Dr. S. Marks and Dr. U. DiGirolami for examination of chickens with lower limb paralysis. We also thank K. Beemon, D. Brown, and C. Martin Stolzfus for critical comments on the manuscript. This work was supported by CA 23086, CA 27223, and CA 32980.
REFERENCES 1. ARRIGO, S., YUN, M., and BEEMON, K., (1987). &-Acting regulatory elements within gag genes of avian retroviruses. Mol. Cell. Ho/. 7(l), 388-397. 2. ARRIGO, S., and BEEMON, K.. (1988). Regulation of rous sarcoma virus RNA splicing and stability. Mol. Cell. Biol. 8(1 l), 48584967. 3. AURIGEMMA, R. E., COMSTOCK, D., and SMITH, R. E., (1989). Persistent viral DNA synthesis associated with an avian osteopetrosis-inducing virus. viroiogy 171, 626-629. 4. AURIGEMMA, R. E., TORGERSEN, J. F., and SMITH. R. E., (1991). Sequences from myeloblastosis-associated virus (MAV-2(O) and UR2AV) involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia. /. viral. 65(l), 23-30. 5. BIZUB, D.. KATZ, R. A., and SKALKA, A. M., (1984). Nucleotide sequence of noncoding regions in Rous-associated virus-2: Comparisons delineate conserved regions important in replication and oncogenesis. J. Viral. 49(2), 557-565.
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6. BROWN, D. W., BLAIS, B. P., and ROBINSON, H. L., (1988). Long terminal repeat (LTR) sequences, env, and a region nearthe 5’ LTR influence the pathogenic potential of recombinants between rous-associated virus types 0 and 1. 1. I/irol. 62(9), 3431-3437. 7. BROWN, D. W., and ROBINSON, H. L., (1988). Influence of env and long terminal repeat sequences on the tissue tropism and avian leukosis viruses. /. Viral. 62(12), 4828-4831. 8. CARLBERG, K., RYDEN, T. A., and BEEMON, K., (1988). Localization and footprinting of an enhancer within the avian sarcoma virus gag gene. /. Viroi. 62(5), 1617-l 624. 9. CULLEN, B. R., SKALKA, A. M., and Ju, G., (1983). Endogenous avian retroviruses contain deficient promotor and leader sequences. Proc. Natl. Acad. Sci. USA 80, 2946-2950. 10. LEAMNSON, R. N., and SHANK, P. R., (1986). Nucleotide sequence comparison of the 3’ regions of avian retroviruses NY203 and NTRE-2. Virology 151, 139-l 45. 11. LINIAL, M. L., and MILLER, A. D., (1990). Retroviral RNA packaging: Sequence requirements and implications. Cuff. Top. A&crobiol. Immunol. 157, 125-l 52. 12. MCNALLY, M. T., GONTAREK, R. R., and BEEMON, K., (1991). Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing. Virology 185, 99-l 08. 13. NECW\MEYER, W. S., and WANG, L-H., (1985). Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family. /. Hrol. 53(3), 879-884. 14. REDDY, E. P., REYNOLDS, R. K., WATSON, D. K., SCHULTZ, R. A.,
15.
16.
17.
18. 19.
20. 21.
22.
871 LAUTENBERGER, J., and PAPAS, T. S., (1983). Nucleotide sequence analysis of the proviral genome of avian myelocytomatosis virus (MC29). Proc. Nat/. Acad. Sci. USA 80, 25002504. ROBINSON, H. L., (1976). Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells. J. Viral. 18, 856-866. ROBINSON, H. L., and MILES, B. D., (1985). Avian leukosis virusinduced osteopetrosis is associated with the persistent synthesis of viral DNA. tirology 141, 130-l 43. ROBINSON, H. L.. REINSCH, S. S., and SHANK, P. R., (1986). Sequences near the 5’ long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis. /. Wol. 59(l), 45-49. SCHWARTZ, D. E., TIZARD, R., and GILBERT W., (1983). Nucleotide sequence of Rous sarcoma virus. Ceil 32,853-869. SHANK, P. R., SCHATZ, P. J., JENSEN, L. M., TSICHLIS, P. N., COFFIN, J. M., and ROBINSON, H. L., (1985). Sequences in the gag-pol5’ env region of avian leukosis viruses confer the ability to induce osteopetrosis. Virology 145, 94-104. SMITH, R. E., (1982). Avian osteopetrosis. Curr. Top. Microbial. lmmunol. 101,75-94. SMITH, R. E., and IVANYI, J., (1980). Pathogenesis of virus-induced osteopetrosis in the chicken. /. Immunol. 125(2), 523530. VOGT, V. M., PEPINSKY, R. B., and SOUTHARD, L. E., (1985). Primary structure of pl9 species of avian sarcoma and leukemia viruses. J. Viral. 56(l), 31-39.
190, 866-87
1 (1992)
5’ Avian Leukosis
H. L.
Virus Sequences
and Osteopetrotic
Potential
R. G. FosrER,t B. P. BLAIS,* S. S. REINSCH,+ M. NEWSTEIN,+ AND P. R. SHANK+
ROBINSON,*!’
Departments of *Pathology and tMolecu/ar Genetics and Microbiology, University Worcester, Massachusetts 0 1655; and +Division of Biology and Medicine, Received
April
13, 1992; accepted
of Massachusetts Brown University, June 29,
Medical Center, 55 Lake Avenue North, Providence, Rhode Island 029 12
1992
Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5’ viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5 sequences that affected osteopetrotic potential resided in an 834-bp region near the 5’ LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA. o 1992 Academic PWSS, I~C.
Avian leukosis virus (ALV) induced osteopetrosis results from the abnormal growth and differentiation of infected osteoblasts (20). The induction of osteopetrosis requires the establishment of a persistent viremia and is associated with the persistent synthesis of viral DNA in bone (3, 16). The persistent synthesis of viral DNA results in the accumulation of 2-20 copies of unintegrated plus integrated viral DNA molecules per bone cell (16, 17). The amount of accumulated viral DNA correlates with both the rapidity and the severity of osteopetrosis induction (16). Several regions of the viral genome determine osteopetrotic potential. The induction of osteopetrosis requires long terminal repeat (LTR) sequences with efficient transcriptional control elements such as those found in the LTRs of exogenousviruses. LTRs of exogenous origin differ in the efficiency with which they induce osteopetrosis. In one series of recombinants, the LTR from Rous-associated virus type 1 was more efficient than the LTR from Prague Rous sarcoma virus-B in causing osteopetrosis (10, 19). In a second series, the LTR of myeloblastosis-associated virus-2(O) had higher osteopetrotic potential than the LTR of UR2-associated virus (4). The LTR of the endogenous Rousassociated virus-O (RAV-0) is substantially less efficient than those of exogenous viruses (9). This LTR cannot support the induction of osteopetrosis (19). At least two regions of the ALV genome outside of the LTR affect osteopetrotic potential. One of these encodes envelope glycoproteins. Envelope glycoproteins that use the subgroup B/E receptor confer higher osteopetrotic potentials than envelope glycoproteins that use the subgroup A receptor (4,.6). This difference presumably reflects differences in the tissue-specific \ ’ To whom 0042.6822/92
reprint
requests
should
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
display or activity of subgroup A and B/E receptors (7). A second region that affects osteopetrotic potential maps to a 1.4-kb region near the 5’ LTR (17). The current study was undertaken to further define the role of 5’ sequences in osteopetrotic potential. To identify 5’sequences that affect osteopetrotic potential, RAV-0 sequences were substituted for homologous 5’ sequences in Br21, a subgroup E virus with high osteopetrotic potential (19). Two independent clones of each of two constructs were constructed using conserved Sac1 and Ball sites to substitute RAV-0 fragments for Br21 fragments (Fig. 1). Plasmids designated pBrl6Os contained RAV-0 sequences from positions 255 to 1089, while pBrl5Os contained RAV-0 sequences from positions 1089 to 1630 of viral RNA (18). The presence of RAV-0 or Br21 fragments in constructs was verified using diagnostic Pstl sites (Fig. 1). gag
__
env
PO1
w LTR
LTR
MA p10 c*
NCPR
c”’
RAV-0
1
I 0
i
I 1
7
I 2
T
i
I 3
i
I 4
I 5
I 6
I 7
kb
FIG. 1. Genomes of RAW0 and Br2 1. LTR. gag, PO/, and env indicate the long terminal repeat regions and sequences encoding the Gag, reverse transcriptase, and envelope proteins respectively. Only the restriction endonuclease sites used for construction and verification of recombinants are indicated. B, Ball; P, Pstl; S, Sacl; X, Xbal. Sites used for verification are indicated with asterisks.
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OSTEOPETROSIS
LOWER LIMB PARALYSIS
GENOME ROOSTER
RAW0
HEN
SEVERE
MILD
SEVERE
0122
o/22
0132
O/32
0122
0132
2l7
2i7
NA
NA
Br21
NA
o/10
Brl3Os
Brl6Os
,gr Is-i LTRc
gag
x
11116
l/15
pal x
env
MILD
NA
o/10
6114
a
NA
NA
NA
NA
NA
0116
NA
5112
2112
NA
NA
5116
6i7
O/7
12l16
0il5
0112
l/12
2l15
Brl40s
BrlliOs
HEN
ROOSTER
O/7
b
l/l2
c
>LTR
FIG. 2. Disease potential of the parent and recombinant viruses. For construction of the Brl3Os and Brl4Os, see Ref (7 7). The Brl6Os and Brl5Os were constructed by substituting the indicated pBr21 fragments into pBrl30 (see text and Fig. 1). Two independently generated DNA clones were used for tests of the Brl30, 140, 150, and 160 recombinants. Tests conducted at different times are tabulated separately. Cases of moderate and severe osteopetrosis are grouped under severe. (a) Four of these six cases of paralysis occurred in birds with severe osteopetrosis. (b) Neither case scored for osteopetrosis. (c) Occurred in the hen with osteopetrosis. NA, not available. Sites for restriction endonucleases used in constructions: B, Ball; P, WI; S, Sacl; X, Xbal.
Plasmid DNAs were transfected into chick embryo fibroblasts (from chicken line 158 evl) for recovery of virus. Virus stocks, designated the Brl5Os and Brl6O.s contained from 1 X 1 O”-1 X 10’ fibroblast-infectious units per milliliter. In viva tests demonstrated that the Brl5Os caused much more rapid and severe osteopetrosis than the Brl6Os (Fig. 2). Tests were initiated by inoculating dayold K28 chicks with 0.2 ml of undiluted virus stocks. At 1 month postinoculation, chicks were bled and tested for viremia using an assay for particulate reverse transcriptase (15). The two test groups exhibited comparable levels of viremia (Fig. 3). By 1 month, Brl50s-inoculated chickens had begun to show signs of osteopetrosis. By 2 months, 74% of the Brl5Os but only 4% of the Brl60s-inoculated birds had developed severe osteopetrosis. Roosters are more susceptible to osteopetrosis than hens (16). In the Brl5Os group, all of the viremic roosters developed moderate to severe osteopetrosis. By contrast, in Brl60s-infected birds, only the roosters with the highest viremias developed moderate to severe disease (Fig. 3).
Brl50s
Brl60s
FIG. 3. Levels of viremia at 1 month postinoculation in Brl50 and Brl60inoculated chicks. Each symbol represents data for one rooster. A, roosters that developed osteopetrosis and paralysis. 0, roosters that developed only osteopetrosis. & rooster that developed only paralysis. 0, roosters that developed mild osteopetrosis or no disease.
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COMMUNICATIONS
. Sac1 . . GAGCTCcAcgGCcgGGggccaagatacCCTaCCGAGaACTCAGAgaGTCGTtGG~GAC~g~~~GCCCgACGACTGAGCaGTCCACccCA~cgtG B~~~:------T-CT--*G--*GCCCAGATAC---A---A-----A-------GA-----T---------GA--GA-----G----------A------~~---G~G*-------GA-----T---------GA--GA-----G----------A------CC---G~cRAV-O:------C-CD--CG--GCCCMGATAA---A----A-----A ev-2 : ev-1: P~C:------T-CT--AG--AGCCCAGATAC---A-----A-------GA-----T---------GA--GA-----G----------A------CC---GCGTMC29:------C-CG--CG--GGGCAAGATAA---A---A-----A-------GA-----T---------GA--GA-----G----------A------CC---GCGTFSV:------C-CG--CG--GGCC~GAT~---------A----------A------CC---GCGTAEV-R:------T-CT--AG--GGCCAAGATAC---A---A-----A-------TA-----T---------GA--GA-----G----------A------AC---GTGCSRA:------C-GG--CC--AGCGACTGACC---G-----A-------GG-----~---------AG--TG-----G----.........----CC---G~A~y73:------C-CG--CG--GGCCAAGATAA---A---A-----A-------GA-----T---------GA--GA-----G----------A------CT---G*C---A-----C-------GA-----T---------GA--GA-----G----------G------CC---ACGTUR2:------T-CT--AG--AGCCAACATAC
*
v
4-
.
atTctGGTCgcCCgG Br21:AT-CT----GC--GWV-O:AT-CC----GC--Tev-2 : ev-1: PrC:AT-CT----GC--GMC29:AT-CT----GC--GFSV:AT-CT----GC--GAEV-R:AT-CC----AC--.SRA:TC-TT----GG--TY73:AT-CC----GC--TURZ:AT-CT----GC--G-
--------------------------------C--G----C---------------------A--------------------------------C--G----C------------------------G-------A--------------------------------
C--G----C-----------------------------------------------C--G----C----------------------------------------------C--A----C----------------------------------------------C--G----C-------------------------------------------G--G----C-------------------------------------------C--G----T-------------------
*
*
. ATaGGGGCCATGtTGTCCCtctTACAAAAgGAAGGGTTGcTTATGTctccCTCAGAcTTATATTCcCCGGGGTCcT~ATCCCAT~CcGCGGCg~ct .
.
Br21:--A---------T------TCT-------G ~V-O:--A---------T-
.
.
.
.
-----------------TCC------C------C---C--------C----
.
554
.
654
-------T--C-----G--A
-----TGT-------G-----------------TCC------C--------C--------C-----------T--C-----G--*
--------T--C-----G--CT
ev-2 : ev-l:--A---------T------TGT-------G -----------------TCC------C------C---C--------C-----------T--C-----G--~ PrC:--A---------T------TCT----G----G----------------TCC------C------C---C--CMC29:--A---------T------TCT----G -----------------Tcc------c------~---c--------A-----------T--c-----A--cA FSV:--A---------T------AGT----G----G-----------------CCT------C--------C--------C-----------T--C-----A--CA AEV-R:--A---------C------TGC----G----G-----------------TCC------C--------C--------C-----------T--C-----G--~ SRA:--A---------T------TGT-----G---G-----------------TCC------T--------T--------C-----------C--T-----G--~ Y73:--A---------C--
----TGT-------G-----------------TCC------C--------C------C---C-----------T--C-----G--~
UR2:--C---------T------TCT-------A-----------------TCC------C--------C---
.
x- *
.
.
.
.
-----C-----------C--T---G--CT
.
*
.
CcCAGCGGGCaATGgTACTTGGgAAATCGGGGAGAGTT~C~GGGGATTGG~TTGGgGGCA~~gGC~CtCGAGA~~CA~~ACAT~GA c--------T---A-------G-------------------------------------G-------A-G-----T-----------------------
Br21::-C--------A---G-------G------------------------------------G-------C-G-----T----------------------RAV-0 _-~ -------------------------------G-------A-G-----T----------------------ev-2 :-C--------A---G-------G-----C--------A---G-------G-------------------------------------G-------A-G-----T----------------------ev-l:PrC :-C--------T---A-------G-------------------------------------G-------A-G-----T----------------------------------------------------------G-------A-G-----T----------------------MC29 :-C--------A---G-------GFsV:-A--------G---G-------G -------------------------------------G-------A-G-----C---------------------------T---G-------G------------------------------------AEV-R:-C--SRA:-C--------A---G------A-------------------------------------G-------A-G-----T----------------------Y73 :-C--------A---G-------G------------------------------------uR2 :-C--------A---G-------G-------------------------------------
XhOI
C-------A-G-----T----------------------G-------A-G-----T----------------------G-------A-A-----C-----------------------
FIG. 4. Sequence data for the Sacl-to-Ball fragments of RAV-0 and Br21 compared with those for other avian leukosis sarcoma viruses. Numbers refer to positions relative to the sequence of Prague Rous sarcoma virus C RNA (18). A consensus sequence is presented above the comparisons. Uppercase letters in the consensus and dashes in viral sequences indicate bases present in all sequenced isolates. Differences between RAV-0 and Br21 are highlighted by asterisks, The positions of restriction endonuclease sites, the splice donor (XD) for subgenomic viral RNAs, and boundaries for MA, ~10, and CA are indicated above the consensus. TSR, a highly conserved translational start region is boxed. The gag enhancer and the NRS regulatory sequences for splicing are bounded by arrows, as indicated. 7 indicates insertions into the sequence of one nucleotide (T) at position 353 in MC29 and of six nucleotides (ACAGCC) at position 883 in SRA and Y73. Data for Prague-C Rous sarcoma virus (PRC), Schmidt-Ruppin-A Rous sarcoma virus (SRA), Avian erythroblastosis virus strain-R (AEV-R), Fujinami sarcoma virus (FSV), and avian sarcoma virus Y73 were obtained from Genbank. Data for myeloblastosis virus MC29 from (74); for UR2, (73), and for ev-1 and ev-2, (22).
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COMMUNICATIONS
E3
869
r
. . . GC~GCAAAGTTTTGGTTGGGATTAGGGGGAGGGAGGGTCG sr~l:---------------------------------------------------------G-------RAv-o:--------------------------------------------------------G-ev-2:--------------------------------------------------------e"-l:--------------------------------------------------------G-----------------A--------------------A-------------------------G-----prc:---------------------------------nc29:--------------------------------------------------------FS":---------------------------------------------------------G-------------AE"-R:--------------------------------------------------------SRA:---------------------------------------------------------G-----------------T--------------------A--~,~:---------------------------------------------------------G-----------------A--------------------G--UR~:---------------------------------------------------------A-----------------A--------------------A---
NRS .
.
754
---------A--------------------A-----------------A-------------------A--G-----------------A-------------------A-------------A-------------------A--G-----------------A--------------------G-----A-------------------G---------.--------A--------------------G~~~
r _
ENHANCER * . . . . -<1 GAGGAagTGGGAGAaACAACTGtGCAGCGAGATgCG~GATGGCGCCgGAGg~acgGCCACACCT~CcG~GGCACATcCTGcTATcAtTGCGG~ S~Z~:-----AG-------A------T----------G-------------G---G--ACG--------------G-------------C-----T----------T----------G-------------G---G--ACG-------------C--------------C---T---T------RAV-O:-----AG-------A--e"-2:-----AG-------A-------T---------G-------------G---G--ACG--------------C--------------C-----T------e"-l:-----AG-------A------C----------G-------------G---A--ATG-------------C--------------C-----G------------T----------G-------------G---G--ACG--------------C--------------C-----T------~~C:-----AG-------AM~~~:-----AG-------A------T----------G-------------G---G--ACT--------------C--------------C-----T-------------A-------------G---G------------C--------------C-----T------. ..------A-------T--FSV:..... AE"-R:-----AA-------G-------T--------G---G------------G---A--ATG-------------C--------------T-----G------C--------------C-----T------SRA:-----GG-------A-------T----------G-------------A---G--GCG-------------Y,3;-----AG-------A------T----------G------------A---G--ACG-------------C--------------C---T---T------"R2:-----AG-------A-------T----------G-------------G---G--ACG --------------C----
. v . . . . r'Pl0 CAGCtatTGGCTGtAAtTGCGcCACaGCCTCgGCcCCTCCTCCtCCtTAtGTGGGGAGT~tTTGTaTCCTTCCCTGGCGGGGGtGGGaGAGCAGCaGgg
854
.
954
.
1054
NRS
ENHANCER
71.
. . . CCaGGGgGgTgAcACacCTcgGGGGGCGGAaCAGcCAAgGGcGGAGCCAgGGCaCgCGGGTCtGgCcCcTGGGCCGGCCCTGACTGACTGGGC~GGaTc
.
.
*
plo<--l-->CA . . aGGGAGGAGCTTGCGAGTACaGGTCCGCCCgTGGTqGCCATGCCTGTAGTGATTAAqACAGAGGGACCCGCcTGGACCCCTCTGGAGCCAaAattgATC B~~~:A-------------------A-------G---G----G--------------------G--------------C------------------A-ATTG--RAV-O:A-------------------A---------G----G--------------------G--------------C------------------A-ATTG--e"-2:A-------------------A---------G----G-----e"-l:A-------------------T---------G----G-----PrC:A-------------------T---------G----G--------------------G--------------T------------------A-ATTG--nC2g:G-------------------T---------G----G--------------------C--------------C------------------A-ATTG--FSV:A-------------------A-------A---A----G--------------------~--------------C------------------A-ATTG--AEV-R:A-------------------T---------G----A--------------------G--------------C------------------G-GGAC--FIG. 4-Continued
.
1154
870
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COMMUNICATIONS
The lower incidence and milder form of Brl60s-induced osteopetrosis differed in its appearance from Brl50s-induced osteopetrosis. Brl5Os caused a generalized increase in the diameter of the leg bones that is characteristic of ALV-induced osteopetrosis. By contrast, Brl6Os induced a lumpy form of osteopetrosis that was manifested as discrete foci of abnormal growth. Brl60s-induced lumps projected from the bone by a few millimeters and had widths of up to 1 cm. This form of disease had been previously noted in Brl30sinfected chickens. The Brl50s-inoculated chicks also developed a high incidence of lower limb paralysis. The appearance of this disability had been previously noted in Br21- and Brl40s-infected chicks (Fig. 2). The lower limb paralysis did not necessarily correlate with the appearance or severity of osteopetrosis (Figs. 2 and 3). Chickens with paralysis did not have joint or hip problems. Serial sections of the brain and the spinal cord of one disabled chicken did not reveal lesions. Thus, the cause-of this disability is not understood. A subgroup A recombinant that induces an intermediate incidence of osteopetrosis (pEU) causes a walking disability associated with ataxia (4). In contrast to the Brl50s-induced paralysis, the ataxia is associated with lesions in the cerebellum (4). pBr21 and pRAV-0 were partially sequenced to identify differences in the fragment that affected osteopetrotic potential. These sequence data are compared with that for other avian leukosis sarcoma viruses in Fig. 4. The 17 bases that differed between RAV-0 and Br21 are indicated with asterisks. Eleven differences were found in noncoding leader sequences. These differences were localized in poorly conserved regions of the leader immediately 3’ to the Sac1 site and immediately 5’ to the highly conserved translational start region (TSR) (5). Positions 271-275 contained changes in three of five bases that were unique to RAV-0. Other differences in the leader represented sequences present in other exogenous viruses. Six differences between RAV-0 and Br21 were present in MA codons. Four of these had no effect on amino acid usage. A codon change at position 569 resulted in a conservative change that is also present in the MA proteins of several exogenous viruses. A codon change at position 623 gave a nonconservative change (a glutamine to a lysine) that is unique to RAV-0. Studies using recombinants between Rous-associated virus-l and RAV-0 have shown that a 375-bp Sacl-to-Xhol fragment from RAV-1 confers osteopetrotic potential (6). Interestingly, this 375-bp region contains all of the unique differences in leader sequences as well as the one nonconservative amino acid difference between RAV-0 and Br21.
Functions encoded in 5’ sequences have not been correlated with osteopetrotic potential. The RAV-0 leader sequence is not deficient for gene expression (9). The leader sequences and MA proteins of osteopetrosis-inducing and non-osteopetrosis-inducing viruses do not affect the efficiency of the formation of infectious particles (unpublished observations). A gag enhancer activity (1, 8) does not distinguish osteopetrosis from non-osteopetrosis-associated sequences (I, unpublished observations). Tests for this enhancer activity have been conducted in a number of cell types including cultured osteoblasts (unpublished observations). The activity of a negative regulator of splicing (2, 12) also does not distinguish osteopetrosis and nonosteopetrosis-inducing infections in fibroblasts and osteoblasts (unpublished observations). In closing, we speculate that the osteopetrosis-enhancing function in 5’ Br21 sequences encodes a function of exogenous but not endogenous viruses. Such is consistent with our identifying osteopetrosis-potentiating functions in 5’ sequences in two series of recombinants between exogenous and endogenous viruses (Fig. 2, Ref. 6). Such is also consistent with recombinants between exogenous viruses failing to reveal a role for 5’ internal sequences in osteopetrosis induction (4). Thus, the 5’ sequences that affect osteopetrotic potential may represent yet another example of a function that has evolved differently in endogenous and exogenous viruses.
ACKNOWLEDGMENTS We are indebted to Dr. S. Marks and Dr. U. DiGirolami for examination of chickens with lower limb paralysis. We also thank K. Beemon, D. Brown, and C. Martin Stolzfus for critical comments on the manuscript. This work was supported by CA 23086, CA 27223, and CA 32980.
REFERENCES 1. ARRIGO, S., YUN, M., and BEEMON, K., (1987). &-Acting regulatory elements within gag genes of avian retroviruses. Mol. Cell. Ho/. 7(l), 388-397. 2. ARRIGO, S., and BEEMON, K.. (1988). Regulation of rous sarcoma virus RNA splicing and stability. Mol. Cell. Biol. 8(1 l), 48584967. 3. AURIGEMMA, R. E., COMSTOCK, D., and SMITH, R. E., (1989). Persistent viral DNA synthesis associated with an avian osteopetrosis-inducing virus. viroiogy 171, 626-629. 4. AURIGEMMA, R. E., TORGERSEN, J. F., and SMITH. R. E., (1991). Sequences from myeloblastosis-associated virus (MAV-2(O) and UR2AV) involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia. /. viral. 65(l), 23-30. 5. BIZUB, D.. KATZ, R. A., and SKALKA, A. M., (1984). Nucleotide sequence of noncoding regions in Rous-associated virus-2: Comparisons delineate conserved regions important in replication and oncogenesis. J. Viral. 49(2), 557-565.
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6. BROWN, D. W., BLAIS, B. P., and ROBINSON, H. L., (1988). Long terminal repeat (LTR) sequences, env, and a region nearthe 5’ LTR influence the pathogenic potential of recombinants between rous-associated virus types 0 and 1. 1. I/irol. 62(9), 3431-3437. 7. BROWN, D. W., and ROBINSON, H. L., (1988). Influence of env and long terminal repeat sequences on the tissue tropism and avian leukosis viruses. /. Viral. 62(12), 4828-4831. 8. CARLBERG, K., RYDEN, T. A., and BEEMON, K., (1988). Localization and footprinting of an enhancer within the avian sarcoma virus gag gene. /. Viroi. 62(5), 1617-l 624. 9. CULLEN, B. R., SKALKA, A. M., and Ju, G., (1983). Endogenous avian retroviruses contain deficient promotor and leader sequences. Proc. Natl. Acad. Sci. USA 80, 2946-2950. 10. LEAMNSON, R. N., and SHANK, P. R., (1986). Nucleotide sequence comparison of the 3’ regions of avian retroviruses NY203 and NTRE-2. Virology 151, 139-l 45. 11. LINIAL, M. L., and MILLER, A. D., (1990). Retroviral RNA packaging: Sequence requirements and implications. Cuff. Top. A&crobiol. Immunol. 157, 125-l 52. 12. MCNALLY, M. T., GONTAREK, R. R., and BEEMON, K., (1991). Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing. Virology 185, 99-l 08. 13. NECW\MEYER, W. S., and WANG, L-H., (1985). Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family. /. Hrol. 53(3), 879-884. 14. REDDY, E. P., REYNOLDS, R. K., WATSON, D. K., SCHULTZ, R. A.,
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22.
871 LAUTENBERGER, J., and PAPAS, T. S., (1983). Nucleotide sequence analysis of the proviral genome of avian myelocytomatosis virus (MC29). Proc. Nat/. Acad. Sci. USA 80, 25002504. ROBINSON, H. L., (1976). Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells. J. Viral. 18, 856-866. ROBINSON, H. L., and MILES, B. D., (1985). Avian leukosis virusinduced osteopetrosis is associated with the persistent synthesis of viral DNA. tirology 141, 130-l 43. ROBINSON, H. L.. REINSCH, S. S., and SHANK, P. R., (1986). Sequences near the 5’ long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis. /. Wol. 59(l), 45-49. SCHWARTZ, D. E., TIZARD, R., and GILBERT W., (1983). Nucleotide sequence of Rous sarcoma virus. Ceil 32,853-869. SHANK, P. R., SCHATZ, P. J., JENSEN, L. M., TSICHLIS, P. N., COFFIN, J. M., and ROBINSON, H. L., (1985). Sequences in the gag-pol5’ env region of avian leukosis viruses confer the ability to induce osteopetrosis. Virology 145, 94-104. SMITH, R. E., (1982). Avian osteopetrosis. Curr. Top. Microbial. lmmunol. 101,75-94. SMITH, R. E., and IVANYI, J., (1980). Pathogenesis of virus-induced osteopetrosis in the chicken. /. Immunol. 125(2), 523530. VOGT, V. M., PEPINSKY, R. B., and SOUTHARD, L. E., (1985). Primary structure of pl9 species of avian sarcoma and leukemia viruses. J. Viral. 56(l), 31-39.