1. health and fitness
2. disease
3. cancer

J.
gen. Virol. (1985), 66, 2057-2063.
Printedin Great Britain
2057
Key words: FeSV/A-MuL V/tran,~'[brmingpr~tein/protein kinase
Immunological and Biochemical Characterization of HZ2 Feline Sarcoma
Virus and Abelson Murine Leukaemia Virus Translation Products
By L Y N N E L E D E R M A N , 1 , 3 t M I T R A C. S I N G H A L , 1 P E T E R B E S M E R , 2"3
E V E L Y N E. Z U C K E R M A N , z W I L L I A M D. H A R D Y , JR 2"3
AND H A R R Y W. S N Y D E R , JRI'3*3~
Laboratories o f 1Viral Oncology and 2 Veterinary Oncology, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, New York, New York 10021 and 3Molecular Biology and Virology
Unit, Cornea University Graduate School o f Medical Sciences, Sloan-Kettering Division,
1275 York Avenue, New York, New York 10021, U.S.A.
(Accepted 23 May 1985)
SUMMARY
The extent of homology between the translation products of the HZ2 strain of feline
sarcoma virus (HZ2-FeSV) and the Abelson murine leukaemia virus (A-MuLV) was
examined immunologically and biochemically. Antiserum prepared against the v-ablencoded determinants of the A-MuLV polyprotein P120ga~-ab~ was also found to
precipitate specifically the 98K mol. wt. HZ2-FeSV protein (P98gag-~bl). The basis for
this immunological crossreactivity was indicated by the findings that the two proteins
had at least six [35S]methionine-containing tryptic peptides and at least eight
[35S]methionine-containing chymotryptic peptides in common. Each of the two
proteins also had tryptic and chymotryptic peptides which were unique. Both proteins
were associated with tyrosyl kinase activities which exhibited some similar
biochemical properties in vitro. However, the HZ2-FeSV-associated activity was much
more sensitive to competitive inhibition by nucleoside and deoxynucleoside
diphosphates than was the A-MuLV-associated activity. These results suggest that,
while the gag-abl translation products of these two independent isolates of
transforming retrovirus are highly related structurally and functionally, the differences
in structure contribute to differences in enzyme activity. Further comparative studies
of these two proteins should play an important role in determining their roles in
induction of two different types of malignancy: lymphosarcoma in the case of the AMuLV protein and fibrosarcoma in the case of the HZ2-FeSV protein.
HZ2 feline sarcoma virus (HZ2-FeSV) is a replication-defective acute transforming retrovirus
obtained from a fibrosarcoma of a young pet cat (Hardy et al., 1982; Besmer et al., 1983). This
virus contains genetic sequences homologous to those of the v-abloncogene (Besmer et al., 1983),
initially described as a component of the Abelson murine leukaemia virus (A-MuLV) genome
(Abelson & Rabstein, 1970a; Goffet al., 1980). Southern blot analysis of mink cell-derived HZ2FeSV DNA and normal mink, cat and mouse D N A using a ~,-32p-labelled v-abl probe (Goffet
al., 1980) and both high and low stringency washing conditions indicated a likely feline origin
for the v-abl sequences in HZ2-FeSV (Besmer et al., 1983). Results also indicated a high degree
of relatedness between the cat- and mouse-derived viral sequences. The v-abl sequences in HZ2FeSV were shown to be linked to feline leukaemia virus (FeLV) sequences in the integrated
t Present address: Department of Biochemistry, Boston University Medical School, 80 East Concord Street,
Boston, Massachusetts 02118, U.S.A.
:1:Present address: Immune Response Program, Pacific Northwest Research Foundation, 1102 Columbia
Street, Seattle, Washington 98104, U.S.A.
0000-6480© 1985 SGM
2058
Short communication
(a)
1
2
3
(b) 1
2
3
(c) 1 2 3
P98gag-ablz>
(d) 4
~
(e)
P 120 gag-abl)"
1
2
3
(f)
1
I
2
5
6
<P95gag-fes
3
i:~i~
N ~
Fig. 1. Reactivity of the translation products of HZ2-FeSV, A-MuLV and GA-FeSV with various
antisera. Aliquots of (a) HZ2-FeSV transformed non-producer CCL64 cells, (b) normal CCL64 cells, (c,
d) GA-FeSV-transformed non-producer CCL64 cells, (e) A-MuLV-transformed NIH 3T3 cells and (f)
normal NIH 3T3 cells, whose proteins had incorporated pH]leucine, were analysed. Immunoprecipitation analysis was performed using 5 p_lquantities of serum from a C57L/J mouse which had regressed a
syngeneic A-MuLV tumour line (anti-AbT), anti-AbT preabsorbed with a predetermined excess of
Moloney MuLV structural proteins (anti-AbT"Us), normal serum from a C57L mouse (nms), serum from
a goat immunized with autocthonous cells transformed with GA-FeSV (anti-GAau0, anti-GA,ut serum
preabsorbed with a predetermined excess of FeLV'structural proteins (anti-GA~bu~)and normal goat
serum (ngs). Immune complexes containing the labelled proteins were collected using SAC (Snyder &
Singhal, 1983). Precipitated proteins eluted from washed SAC were separated in slab gels with a
gradient of 7-5 to 12.5~ acrylamide and visualized by autoradiography of dried gels. Proteins coprecipitating with anti-ABT serum are shown in lanes 1, those with anti-AbT~us are in lanes 2, those
with nms are in lanes 3, those with anti-GA:,u~ are in lane 4, those with anti-GA~b~ are in lane 5 and those
with ngs are in lane 6.
genome (Besmer et al., 1983). Transcription of those sequences in infected CCL64 mink cells
gives rise to two RNA species. One of the RNAs (6.5 kb) contains v-abl and FeLV gag and env
gene sequences but lacks most of t h e p o l sequences. This RNA is presumed to be the full-length
genome of HZ2-FeSV. The other RNA is 2-3 kb and only contains env sequences, suggesting
that it is a subgenomic mRNA. The translation product of the HZ2-FeSV genome was initially
investigated by immunoprecipitation analysis of extracts of metabolically labelled infected
mink cells using monospecific antisera to FeLV structural proteins (Besmer et al., 1983). The
only product identified was one of 98 000 (98K) molecular weight. This protein, referred to as
P98, was detected with antisera specific for FeLV pl 5, p l2 and p27 but not by antisera against
FeLV gp70. A protein kinase activity was found to be associated with P98 in specific immune
complexes bound to Staphylococcus aureus Cowan I (SAC). While these studies were consistent
with the possibility that P98 is a gag-abl fusion protein and, by implication, the transforming
protein of HZ2-FeSV, this result was not rigorously established. Data are presented here which
confirm and extend the above results by analysis of the protein products of HZ2-FeSV and AMuLV.
The immunological relationship of the HZ2-FeSV-encoded protein to that encoded by AMuLV was investigated by immunoprecipitation of the proteins from virus-transformed cell
lines using antibodies prepared against the sarcoma virus-specific domains of the A-MuLV
protein. C57L anti-AbT serum, produced in mice which regressed a syngeneic A-MuLV tumour
line, was used (Witte et al., 1979). In addition to antibodies against transforming virus-specific
domains, this serum also contained antibodies to Moloney MuLV proteins. The latter class of
antibodies was absorbed out of some aliquots of anti-AbT serum with predetermined excess
amounts of non-ionic detergent-disrupted Moloney MuLV prior to their use. Absorbed antiAbT, unabsorbed anti-AbT or normal serum from a C57L mouse were then mixed with aliquots
of lysates of cells whose proteins had been metabolically labelled with [3H]leucine. The resulting
immune complexes were collected by adsorption to SAC and the associated labelled proteins
Short communication
(a)
(b)
(e)
2059
(a9
P98gag-abl
92
68
46
30
Fig. 2. Reactivity of the HZ2-FeSV translation product with antisera raised in goats against FeSVgagJes and gagz/ms polyproteins. Aliquots of HZ2-FeSV-transformed non-producer CCL64 cells, whose
proteins had been labelled with [3H]leucine, were immunoprecipitated with antiserum against (a) the
SM-FeSV protein (anti-SMppt), (b) anti-SMppt absorbed with FeLV structural proteins, (c) antiserum
against the GA-FeSV protein (anti-GA:~oT)and (d) anti-GA,,u~absorbed with FeLV structural proteins.
Precipitated proteins were analysed by SDS-PAGE as described in the legend to Fig. 1. Mol. wt. x
10-3 is indicated.
were analysed by polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol and
SDS (SDS PAGE). A-MuLV Pl20g~g -~bl was immunoprecipitated from a lysate of A-MuLVtransformed N I H 3T3 cells (Scher & Siegler, 1975) with anti-AbT serum but it was not
precipitated with normal C57L serum (Fig. 1). In CCL64 mink cells transformed with HZ2FeSV (derived in this laboratory), P98 gag-abjwas also specifically recognized by anti-AbT serum.
The recovery of the FeSV protein by immunoprecipitation was similar to the recovery of the AMuLV protein, suggesting that the antiserum recognized highly related sequences in the
respective transforming virus-specific domains. In contrast, the Gardner Arnstein (GA) FeSV
translation product P95 gag-fes,also expressed in a CCL64 cell line (Henderson et al., 1974, Sliski
et al., 1977; Stephenson et al., 1977), was not precipitated by anti-AbT but was recogniZed by
antibodies to fes-encoded determinants (Barbacid et al., 1980). A second protein of
approximately 90K mol. wt. in both normal and transformed mink and mouse cells was nonspecifically precipitated with both anti-AbT and normal C57L serum. This protein was not coprecipitated with the goat antiserum used in this experiment.
The crossreactivity of P98 ~ag-~blwith antisera raised against other FeSV-encoded polyproteins
was also assessed by radioimmunoprecipitation analysis. Results shown in Fig. 2 indicated that
antisera raised against FeSV gag-fes and gag-ires proteins (Barbacid et al., 1980) effectively
precipitated the HZ2-FeSV protein. However, absorption analysis suggested that these
reactions were only mediated by the antibodies in test sera with specificity for the gag-encoded
portions of these proteins and not by antibodies specific for v-fes- and v-ires-encoded
determinants.
The basis for the strong immunological crossreactivity displayed by the HZ2-FeSV and AMuLV translation products was revealed by tryptic and chymotryptic peptide analysis of the
proteins. The procedure published by Van de Ven et al. (1980) was used. HZ2-FeSV P98 gag-abj,
labelled with [35S]methionine and purified from transformed CCL64 cells by immunoprecipita-
2060
Short communication
Fig. 3. Comparisons of [35S]methionine-containing tryptic and chymotryptic peptides of(a, d) HZ2FeSV P989,9-~b~and (b, e) A-MuLV P 120g"9-abland (c,f) mixtures. The polyproteins were obtained from
transformed ceils by immunoprecipitation with specific antiserum followed by purification by SDSPAGE. They were subjected to enzyme digestion and peptide analysis as described by Van de Ven et aL
(t980). Digests were applied to cellulose thin-layer chromatography plates at the bottom left corners.
Electrophoresis, from left to right, was followed by ascending chromatography. The radiolabelled
peptides were visualized by autoradiography of the dried plates. Tryptic peptides of the proteins
individually and in a 1:1 mixture are shown in (a) to (c). Chymotryptic peptides of the proteins are
shown in panels (d) to (1).
tion and S D S - P A G E , was composed in part of eight well-resolved methionine-containing
tryptic peptides (designated 1 to 8 in Fig. 3a). A - M u L V P120 g~g~bl, labelled with
[3SS]methionine and purified from transformed N I H 3T3 cells in the same manner, displayed
at least seven methionine-containing tryptic peptides (designated a to g in Fig. 3 b). W h e n a
mixture of the two proteins was analysed, nine methionine-containing tryptic peptides were
resolved (Fig. 3c). Six peptides (designated 2b, 3c, 4d, 6e, 7f and 8g) a p p e a r e d to be shared by
both viral proteins while peptides 1 and 5 appeared to be unique to H Z 2 - F e S V P989ag-abl and
peptide a appeared to be unique to A - M u L V P120 gag-abl.
Analysis of H Z 2 - F e S V P989ag-abj digested with ~-chymotrypsin revealed more than ten
methionine-containing peptides (Fig. 3d). The ten peptides which were repeatedly resolved
were designated 1 to 10. At least nine methionine-containing chymotryptic peptides were
detected in A-MuLV PI20 g~g-~b~(Fig. 3e). These fingerprints were complicated by the presence
of numerous less well-resolved peptides. However, there were some striking regions o f
homology. The characteristic doublets formed between peptides 1-2 and 3-4 in the P98 gag-abl
fingerprint were displayed as peptides a - b and c - d in the P120 gag-ab~ fingerprint and as comigrating peptides in analysis of the mixture (Fig. 3f). Also, the linear arrangement of peptides
4, 5 and 7, 8 and 10 correlated with peptides c, e and g, h and i, respectively (Fig. 3f). Peptides 6
and 9 appeared to be specific for H Z 2 - F e S V P989ag-ab] and peptide f appeared to be specific for
A - M u L V P 120 gag-abl.
The tyrosyl protein kinase activities associated with expression of the H Z 2 - F e S V and AMuLV proteins in cells were compared. Sefton et al. (1981) demonstrated that the proteins of
N I H 3T3 cells infected with and transformed by A - M u L V contained seven- to 12-fold higher
levels of phosphotyrosine than did proteins of non-transformed N I H 3T3 cells. To determine
whether this property was also associated with infection of cells by HZ2-FeSV, transformed
Short communication
2061
CCL64 cells and uninfected CCL64 cells were incubated with 32p and the proteins were
extracted and subjected to phosphoamino acid analysis (Hunter & Sefton, 1980). The amount of
phosphotyrosine in CCL64 cell proteins, as a percentage of the total detectable phosphoamino
acids, was 0.02 ~, while the amount of phosphotyrosine in HZ2-FeSV-transformed CCL64 cells
was 0"22~o, an ll-fold higher level (data not shown). Thus, elevated levels of tyrosine
phosphorylation were also found in vivo in HZ2-FeSV-transformed cells.
In vitro, immunoprecipitates containing A-MuLV P 1200ao-ab~were shown to contain a tyrosyl
protein kinase activity, presumed to be associated directly or indirectly with the elevated levels
of phosphotyrosine in vivo (Van de Ven et al., 1980; Witte et al., 1980; Sefton et al., 1981). This
activity results in the transfer of label from [y-32P]ATP to the viral polyprotein and, in some
cases, to the heavy chain of IgG in the SAC-bound immune complexes. Immune complexes
containing HZ2-FeSV P989ag-ab~also displayed an autophosphorylating activity (Besmer et al.,
1983). The phosphoamino acid content of P98 gag-ablphosphorylated in vitro was determined by
isolating the labelled protein from preparative SDS-polyacrylamide gels, subjecting it to
hydrolysis in the presence of 6 M-HCI and analysing the products by two-dimensional
electrophoresis on cellulose plates followed by autoradiography (Hunter & Sefton, 1980). As
anticipated, most of the 23p incorporated into P989ao-ab'corresponded to [32p]phosphotyrosine
whereas only trace amounts of radioactivity migrated with marker phosphoserine and
phosphothreonine (data not shown).
Since the biochemical properties of the A-MuLV-associated kinase had already been
catalogued (Van de Ven et al., 1980; Witte et al., 1980), it was of interest also to analyse the HZ2FeSV P989ag-abZ-associated kinase activity further. This was accomplished by analysing the
associated activity under different experimental conditions in immunoprecipitates containing
the protein. This activity was found to prefer Mn 2÷ and Co 2+ over Mg 2÷ as the divalent cation,
while Ca z+ failed to initiate any activity in vitro. The enzyme bound G T P as well as ATP,
although with much less efficiency. Maximal transfer of 7-32p from ATP to the polyprotein was
completed in less than 1 min at 0 °C as well as at 30 °C. The optimal pH for the reaction was near
7.0. These properties closely resembled those reported to be associated with the A-MuLV kinase
and they established that HZ2-FeSV P98 gag-ab~was related functionally as well as structurally to
A-MuLV P1200~'0-abl.
The effects of nucleoside tri-, di- and monophosphates on the phosphorylation activities
found in immune complexes containing HZ2-FeSV P98 gag-abland A-MuLV P 120gag-"b~were also
evaluated using [7-3zp]ATP as the phosphate donor. For comparison, analysis of complexes
containing GA-FeSV P95 g"gt~s was also performed. Both the HZ2-FeSV and the GA-FeSV
proteins were obtained from transformed CCL64 cells. The results of this analysis are shown in
Table 1. None of the protein kinases was inhibited by addition of excess CTP, CDP, dCDP,
AMP and GMP. Inclusion of cAMP or c G M P did not inhibit or stimulate the enzyme activities.
Only ATP and higher levels of ADP and dADP produced inhibition of P95gag-feS-associated
protein kinase activity. In the case of HZ2-FeSV P98 g~-"bl, both ATP and G T P produced
marked competitive inhibition of the protein kinase activity although G T P was less effective
than ATP. Higher levels of ADP, dADP, G D P and d G D P were also inhibitory. In contrast,
only ATP and, to a lesser extent, G T P were effective inhibitors of P120ga~-ab~-associated protein
kinase activity.
The lack of total homology between the translation products of HZ2-FeSV and A-MuLV is
most clearly demonstrable in the different biochemical properties of the associated protein
kinase activities. Although both enzymes share important primary characteristics (tyrosine
specificity, preferred use of Mn z+ as the divalent cation) the HZ2-FeSV kinase activity was
much more sensitive to competitive inhibition by nucleoside and deoxynucleoside diphosphates
than the A-MuLV activity. Both strains of virus transform cells as a consequence of their
acquisition of selected c-abl genetic sequences from their respective species of origin. Thus, the
differences in the enzymic properties could be a reflection of differences between cat and mouse
c-abl sequences which are not relevant to the primary action of the gene products in vivo.
Alternatively, sequences external to the v-abl-encoded portions of these proteins could be
responsible. The v-abl-encoded region of P120 gag-"bl is approximately 90K mol. wt. in size
Short communication
2062
Table
1. Inhibition of protein kinase activity associated with transjbrming proteins o f three
retroviruses*
Viral protein
Concentration of inhibitor
Inhibitor
ATP
GTP
ADP
GDP
dADP
dGDP
AMP, CTP, cAMP, CDP,
cGMP, dCDP, GMP
GA-FeSV
HZ2-FeSV
A-MuLV
P959ao -fes
p98 ~ag-abl
P 1200ao-abl
lO~tM IO0~M 1 mM
lOlaM 100 ~tM 1 mM
lO~tM IO0~M 1 mM
>90
<10
56
<10
40
<10
<10
>90
68
65
44
64
55
<10
>90
80
<10
<10
<10
<10
<10
>90
<10
65
<t0
69
<10
<I0
>90
<10
>90
<t0
>90
<10
<10
>90
>90
76
54
86
65
<10
>90
>90
80
70
>90
76
<10
>90
>90
<10
<10
<10
<10
<10
>90
>90
<10
<10
<10
<10
<10
* The protein kinase activities associated with the viral polyproteins in immune complexes were assessed as
described by Snyder & Singhal (1983). SAC-bound immune complexes containing the appropriate viral
polyprotein were resuspended in 20 ~tl of a reaction buffer containing 0.4 rtM [~'-3-'P]ATP (3000 Ci/mmol, New
England Nuclear) and incubated at 30 °C for 10 min. After termination of the reaction, SAC eluates were analysed
for trichloroacetic acid-precipitable radioactivity. In some experiments various ribonucteoside and deoxyribonucleoside phosphates were also present in the reaction mixtures in final concentrations of 10, 100 and 1000 ~tM.
(Baltimore et al., 1979). T h e extent o f P98 g"g-"b~ that is e n c o d e d by v-abl sequences has not yet
been d e t e r m i n e d . It is possible that the presence o f fewer or additional gag d e t e r m i n a n t s on the
a m i n o end of H Z 2 - F e S V P98g~g -ab~a n d / o r some partial pot or env sequences on the carboxyl end
could h a v e effects on the tertiary structure o f the polyprotein and on nucleoside p h o s p h a t e
binding.
E v e n though H Z 2 - F e S V and A - M u L V had both a c q u i r e d c-abl genetic sequences the extent
of h o m o l o g y between the translation products of the two viruses could not be p r e d i c t e d for
several reasons. First, the viruses were originally isolated from different t u m o u r types as well as
f r o m different species: A - M u L V was d e r i v e d from a l y m p h o s a r c o m a of a steroid-treated
B A L B / c m o u s e infected with M o l o n e y M u L V (Abelson & R a b s t e i n , 1970a) while H Z 2 - F e S V
was d e r i v e d from a naturally occurring fibrosarcoma in a pet cat infected w i t h F e L V (Besmer et
al., 1983). Second, while A - M u L V t r a n s f o r m s b o t h h a e m a t o p o i e t i c (phenotypically pre-B) cells
and fibroblasts, it induces only leukaernia in vivo (Abelson & R a b s t e i n , 1970b; R a b s t e i n et al.,
1971 ; Scher & Siegler, 1975" B a l t i m o r e et al., 1979; Cook, 1982; R o s e n b e r g , 1982). In contrast,
H Z 2 - F e S V has only been shown to induce fibrosarcomas in vivo (Besmer et al., 1983). T h i r d ,
while the relative contributions of helper virus-specific and abl-specific sequences to A - M u L V
are k n o w n , those associated w i t h H Z 2 - F e S V h a v e not yet b e e n d e t e r m i n e d .
W e conclude, therefore, that while the gag-abl translation products of these two i n d e p e n d e n t
isolates of t r a n s f o r m i n g retrovirus are highly related structurally and functionally, the
differences in structure contribute to m e a s u r a b l e differences in e n z y m e activity. F u r t h e r
c o m p a r a t i v e studies of these two proteins should play an i m p o r t a n t role in ultimately
u n d e r s t a n d i n g their roles in two different types o f malignancy.
Antisera to the FeSV gag-fes and gag-fms proteins were generously provided by M. Barbacid of the National
Cancer Institute. Antiserum to the A-MuLV gag-abl protein was generouslY provided by N. Rosenberg, Tufts
University School of Medicine. Excellent technical assistance was provided by R. Markovich. H.W.S., Jr is a
Scholar of the Leukemia Society of America. This work was supported by grants CA-16599 and CA32926 and core
grant CA-08748 from the National Institutes of Health, U.S.A.
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(Received 26 November 1984)