an interesting murine model to study retrovirus-induced immunosuppression on the basis of its ..... FIS-2 and of SFFVp showed that one direct repeat is com-.
Vol. 68, No. 11
JOURNAL OF VIROLOGY, Nov. 1994, P. 6976-6984
0022-538X/94/$04.00+0 Copyright ©) 1994, American Society for Microbiology
Molecular Cloning and Characterization of an Immunosuppressive and Weakly Oncogenic Variant of Friend Murine Leukemia Virus, FIS-2 HONG YAN DAI,l* ARILD FAXVAAG,2t GUNN IRENE TROSETH,' HARALD AARSET,3 AND ARE DALEN4 Unigen Center for Molecular Biology,' and Institute of Cancer Research,2 University of Trondheim, N-7005 Trondheim, Department of Pathology,3 and Department of Microbiology,4 Trondheim Regional Hospital, Trondheim, Norway Received 22 March 1994/Accepted 10 August 1994
The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.
However, the mechanism by which FV induces immunosuppression still remains obscure. One of the reasons is that FV-induced immunosuppression is a complex phenomenon involving many different cell types of the immune system, including B and T lymphocytes, myeloid cells, and erythroid cells. The potent leukemogenicity of FV has also complicated the study of this phenomenon because of the early infiltration of the leukemic cells. We recently described the isolation of a weakly leukemogenic, but relatively strongly immunosuppressive variant of FV from the T helper cells of the FV-infected adult NMRI mice (15). This variant exhibits immunosuppressive properties similar to those of FV but is diminished in its ability to induce acute erythroleukemia. The adult NMRI mice infected by this variant developed a disease which had many of the features of AIDS, which is characterized by polyclonal T- and B-cell activation, hyperglobulinemia, and abnormal production of different cytokines (16, 17). A wasting disease, with some resemblance to what is seen in patients with AIDS, that was characterized by weight loss, atrophy of thymus and lymph nodes, and renal disease was observed in some terminally infected mice. Therefore, this variant was initially designated Fd-MIV for Friend-derived murine immunodeficiency virus (15). Since the name MIV could be easily misunderstood as a murine retrovirus related to lentivirus, in the present report we rename Fd-MIV with the designation FIS variant for Friend immunosuppressive variant. We reported previously that this FIS variant was a mixture of at least two viral components, a replication-competent F-MuLV-related viral component and a mink cell focusforming virus-related defective virus (15). In the present study, we have biologically purified the variant. We show that a
Immunosuppression is a phenomenon frequently associated with retrovirus infections. One of the most-used animal model to investigate this phenomenon has been the Friend murine leukemia virus complex (FV). This complex is composed of a replication-competent Friend murine leukemia helper virus (F-MuLV) and a replication-defective, acutely transforming spleen focus-forming virus (SFFV) (48). In adult mice of susceptible strains, it induces rapid splenomegaly with polyclonal erythroid proliferation and ultimately erythroleukemia (48). Profound immunosuppression was observed early in the mice after infection. For example, the specific response of B cells to certain antigens such as sheep erythrocytes (SRBC) is depressed severely both in vivo and in vitro from only a week after mice become infected with FV (1, 3, 13). The ability of T cells to mediate cytolysis of allogenic target cells is reduced (38), and the generation of cytotoxic T cells is impaired from 2 weeks onward after infection (18). Also, the number of T helper cells is drastically decreased from that time (28). The functions of other immunocytes, such as macrophages, have also been reported to be inhibited (28). Therefore, both the humoral antibody response and the cell-mediated response are remarkably affected by FV infection. Many previous studies have indicated that the retrovirus transmembrane protein TM itself plays a direct role in retrovirus-induced immunosuppression (8, 47). The genotype of the mouse major histocompatibility complex was shown to have strong influence on FV-induced immunosuppression (37).
* Corresponding author. Phone: 47 73 598690. Fax: 47 73 598705. t Present address: Department of Rheumatology, Trondheim Re-
gional Hospital, Trondheim, Norway. 6976
FIS-2 VARIANT OF F-MuLV
VOL. 68, 1994
biological clone, FIS-2, which was obtained by end point dilution is an F-MuLV-related virus and is responsible for the disease induced by the FIS variant. We also describe the molecular cloning and characterization of FIS-2. Restriction mapping and nucleotide sequence analysis of FIS-2 show a high degree of homology between FIS-2 and the prototype F-MuLV clone 57. A striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat (LTR). Multiple point mutations are also found in the structure genes. Our results demonstrate that these minor sequence differences can generate a virus variant with different biological properties. MATERIALS AND METHODS Cell lines and virus. Mouse NIH 3T3 fibroblasts were used in the transfection studies and end point dilution cloning. They were cultured in Dulbecco modified Eagle medium supplemented with 10% newborn calf serum, 2 mM L-glutamine, and 0.05 mg of gentamicin per ml. The F-MuLV clone 57 used in these studies was obtained from the NIH 3T3 cells transfected with plasmid 2-lalc (25), which contained the molecularly cloned F-MuLV genome. The primary stock of the FIS variant was maintained in NIH 3T3 cells (15). Biologically cloned FIS-1 was obtained by end point dilution of the primary stock of the FIS variant. Briefly, NIH 3T3 cells cultured in 24-well plates were infected with various dilutions of the primary stock. The wells containing infected cells were detected by reverse transcriptase activity in the cell-free supernatant (19). The clone FIS-1 was selected from the well, which showed low reverse transcriptase activity after infection with the terminally diluted virus. The biological clone FIS-2 was obtained by end point dilution of the supernatant of the NIH 3T3 cell culture, which was established by coincubation with the cell-free spleen homogenate from the mice suffering from wasting disease. Two rounds of end point dilution were performed for isolation of clone FIS-1, and one was performed for isolation of clone FIS-2. Preparation and analysis of unintegrated viral DNA. A modified Hirt procedure was used for the preparation of unintegrated viral DNA (5). For Southern blot analysis, the viral DNA of F-MuLV clone 57 was isolated from the NIH 3T3 cells transfected with plasmid 2-lalc. The viral DNAs of FIS-1 and of FIS-2 were isolated from the NIH 3T3 cells productively infected with the biological clones of FIS-1 and of FIS-2, respectively. The Southern blot analyses of the viral DNA were performed according to standard techniques (23). The plasmid lalc-2 and an SacII fragment containing the most of the F-MuLV envelope gene were used as probes in the Southern blot analyses
(25). For the molecular cloning of FIS-2, the unintegrated viral DNA was prepared from NIH 3T3 cells newly infected with the biologically cloned FIS-2. The molecular cloning of FIS-2. The schematic strategy of the molecular cloning of FIS-2 is shown in Fig. 1A. The viral DNA was digested with the restriction enzyme EcoRI and was separated by 1% agarose gel electrophoresis. The gel slice around the fragment size of the appropriate 9 kb was excised, and DNA was eluted using the SpinBind TM DNA extraction unit (FMC Bioproducts). The removal of the viral DNA from the gel was confirmed by Southern blot hybridization with the F-MuLV env probe. The DNA fragment was concentrated by ethanol precipitation and was ligated with pUC19, which had been digested with EcoRI and treated with calf intestinal alkaline phosphatase. The ligation was performed with T4 DNA ligase (New England Biolabs) at room temperature
6977
overnight. The ligation mixture was used to transform competent Eschenichia coli DH-5oa cells (6). The white transformants were selected on the Luria-Bertani (LB) plates containing 40 ,ug of ampicillin per ml, 0.5 mM isopropyl-p-D-thiogalactopyranoside, and 40 ,ug of 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside (X-Gal) per ml. The positive clones with the viral DNA as insert were identified by colony filter hybridization (45) with the F-MuLV env probe (25). The clones were given the designation pUC-FIS-2. The plasmid pUC-FIS-2 was further digested with ClaI and EcoRI, and the fragment containing the LTR, gag, and the partialpol gene was isolated from the agarose gel and ligated into pBR322, which had been digested with EcoRI and ClaI. The clone was named pBR-FIS-2-lg. The construct with the permuted structure of FIS-2 containing both 5' and 3' LTRs, pBR-proFIS-2, was achieved by inserting the EcoRI viral fragment isolated from pUC-FIS-2 into pBR-FIS2-1g. The correct orientation was confirmed by restriction enzyme analysis. E. coli HB101 was used for the transformation of pBR-proFIS-2. DNA transfection of NIH 3T3 cells. NIH 3T3 cells were transfected with the DNA of plasmid pBR-proFIS-2 by the calcium phosphate precipitation method (22). The production of infectious virus was confirmed by the presence of reverse transcriptase activity (19) in cell-free culture fluids. The DNA used for transfection was isolated by cesium chloride gradient
centrifugation.
Nucleotide sequence analysis. DNA sequencing was done using the Sequenase version 2.0 kit according to the instructions of the manufacturer (U.S. Biochemical Corp.). The strategy of directed sequencing with progressive oligonucleotides was applied. The plasmid pBR-FIS-2-lg was used to sequence the LTR region and the gag gene. The plasmid pUC-FIS-2 was used to sequence the env gene. With the exception of the commercially purchased Hindlll primer (New England Biolabs) that was used at the beginning of the sequencing, the other oligonucleotides used in the sequencing were synthesized by our own DNA synthesis service. The
nucleotide sequence of the F-MuLV clone 57 was obtained from GenBank (accession no. X02749). Primary antibody response in adult NMRI mice. The retrovirus-infected and the uninfected control mice were immunized with 108 SRBC per 0.2 ml of phosphate-buffered saline intravenously. After immunization for 4 days, splenocytes were isolated, and the number of B cells producing anti-SRBC antibody per 104 and 105 splenocytes was measured by the modified Cunningham technique as described previously (15). Mice, disease induction, and classification of disease. Female adult NMRI mice (age 6 weeks) with average weights of 20 g at arrival (Bomholdtgaard, Breeding Research Center, Rye, Denmark) were fed with food pellets and water ad libitum. The adult mice were inoculated with 104 infectious units intraperitoneally for the study of disease induction. Female NMRI mice were obtained at 2 weeks of gestation, and the newborn mice were inoculated with 3 x 105 infectious units intraperitoneally within 36 h after birth. The titer of F-MuLV clone 57 was determined by the XC plaque assay. Mice were sacrificed by cervical dislocation when the overt signs of the diseases began to show. Autopsy was performed. Lymphoid organs and kidneys were weighed. The tissue sections of representative cases from each group were microscop-
ically investigated.
Nucleotide sequence accession numbers. The nucleotide sequences of the LTR region and the gag and env genes of FIS-2 were submitted to the EMBL data library. The accession numbers of the LTR region, the gag gene, and the env gene are Z35111, Z35110, and Z35109, respectively.
6978
J. VIROL.
DAI ET AL.
(A)
Clal
C(Is t
EEm-
~~~~Viral DNJAisdated by Hirt extraction
J EDORt
|Di"sten of
Viftl CM with EeR!
CL.'
EcoRI
ClsI
Cial
EcdUl
t
(
.EccRI
doRI
pUC-FS-2
pBR-FIS-2-g
\ EcaRI
ccRI
\s,c_Clot
provi rus (B)
P
K
P
>fHP PH
Pv
I 11
E
Pv
~~I
H
I.
SP C
S
UlI R
Po]
gag
env
LTR
K K
M[½
~~ b
I I II
Ul u~~FVb5t R\U
LTR
K
U5
F-MuLV clone 57
U3T ~~~~~~~~~~~~LTR K K
P PH
I I I1 FLo-
Pv
EK II
Pv
U3 R U5
H
S
S SP C t ~II
FIS-2
U3 R U5
1 kb FIG. 1. (A) Schematic diagram for the molecular cloning of FIS-2. The restriction enzyme sites used for cloning are indicated. The procedure is described in more detail in Materials and Methods. (B) Comparison of the restriction enzyme maps of FIS-2 versus F-MuLV clone 57. The restriction enzyme map of F-MuLV clone 57 is derived from the published nucleotide sequence. Abbreviations: C, ClaI; E, EcoRI; K, KpnI; H, HindIII; P, PstI; Pv, PvuII; S, SacIl.
RESULTS FIS-2 is a replication-competent virus. Our early study showed that the primary stock of the FIS variant contained a mixture of at least two viral components, the replicationcompetent F-MuLV-related virus and the replication-defective mink cell focus-forming virus related virus (15). Since the defective viral RNA was not detected in the mice which finally developed wasting disease after infection with the FIS variant, we assumed that the replication-competent F-MuLV-related virus might be responsible for the disease. To prove this, we started to purify single biological clones from the primary stock of the FIS variant and from the supernatant of an NIH 3T3 cell culture, which was established by coincubation with the cellfree spleen homogenate from the mice suffering from wasting disease. Two single clones of the replication-competent viruses FIS-1 and FIS-2 were isolated by end point dilution. The unintegrated viral DNAs from the cultures producing the clones of FIS-1 and of FIS-2 were isolated. Southern blot analyses showed that the defective viral component was eliminated during the procedure of end point dilution (data not shown). The unintegrated viral DNAs of both clones were digest-
ed with several restriction enzymes, EcoRI, KpnI, PstI, and HindIII. The fragments were analyzed using probe containing the whole F-MuLV clone 57 genome. The analysis showed that the two clones had identical restriction enzyme patterns, which indicates the identity of the clones. The clone FIS-2 was further molecularly cloned into pBR322 as described in Fig. 1A and Materials and Methods. To show that the molecularly cloned FIS-2 is infectious in NMRI mice, the adult mice were inoculated with the supernatant of NIH 3T3 cell culture, which was transfected with pBR-proFIS-2 (Fig. 1). Three days after inoculation, the presence of infectious viral particles in the blood and in the cell-free spleen homogenate and the presence of infected splenocytes were demonstrated by positive reverse transcriptase activity in the cell culture fluids of NIH 3T3, which were coincubated with serum, cell-free spleen homogenate, and splenocytes from infected mice. In contrast, the NIH 3T3 cells coincubated with serum, cell-free spleen homogenate, and splenocytes from uninfected NMRI mice gave negative reverse transcriptase activity. Molecular characterization of FIS-2. To further characterize the FIS-2 genome, we developed a restriction enzyme map
FIS-2 VARIANT OF F-MuLV
VOL. 68, 1994
the infected mice related to that of uninfected mice is shown in Fig. 2. The result shows that the molecularly cloned FIS-2 is able to suppress primary antibody response to SRBC in infected mice as strongly as the primary stock of the FIS
150
-L 0 cD
6979
O 100
variant (15). Not only was the suppressive activity of FIS-2 higher than that of F-MuLV clone 57, but the onset of this
F-MuiLV clone 57
-
Eo
activity was also much faster and more efficient than that of F-MuLV clone 57. To study the consequence of this strong and persistent activity suppressing the primary antibody response, the adult NMRI mice inoculated with FIS-2 or with F-MuLV clone 57 were under observation until terminal disease developed (Ta-
0 0 0
a, 50
2 -F -2_
ble 1). All mice infected with F-MuLV clone 57 developed tumors, including erythroleukemia, lymphoma, and other un-
130
100
50
C
Days after virus infection
specified neoplastic diseases after long latency. Most of the F-MuLV clone 57-infected mice showed advanced disease after infection for about 600 days, at the time when the
FIG. 2. Primary antibody response against SRB C in adult NMRI mice infected by FIS-2 and F-MuLV clone 57. The B cells producing anti-SRBC antibody were counted as plaque-forr ning cells by the modified Cunningham method. Each value in the cuirve represents the average number of plaque-forming cells from five nnice.
uninfected control mice also started to die (data not shown). This suggested that aging was
one
of the factors that affect the
mortality of F-MuLV clone 57-infected mice in this experi-
ment. Interestingly, one-third of the FIS-2-infected mice developed malignant lymphoma and advanced kidney disease. Histopathological analyses showed that the glomeruli of kidney were enlarged. The same kidney changes were described previously (15). Further study showed deposition of immune complex in renal glomeruli as in the cases of glomerulonephritis (data not shown). Such renal disease was not observed in F-MuLV clone 57-infected mice, even after an extensive observation period. As FIS-2 induced suppression of the primary antibody response more rapidly and efficiently in adult mice than F-MuLV clone 57, it also induced disease more rapidly than F-MuLV clone 57 (Fig. 3A). These results indicate that FIS-2 is a more pathogenic variant of F-MuLV in adult mice. A group of FIS-2-infected adult mice were also sacrificed before any signs of clinical disease were observed. Autopsy showed slight enlargement of lymph nodes and spleen. Microscopic analyses showed follicle hyperplasia as described previously (15). An expansion of the sinus system with macrophages occurred in some of the lymph nodes. No signs of malignant lymphoma were observed in these slightly enlarged lymph nodes, even after infection for more than a year. Since such enlargement of lymph nodes was not detected in the majority of mice infected with F-MuLV clone 57, this result indicates that lymph nodes are more affected by infection with FIS-2 than by infection with F-MuLV clone 57. Since the oncogenicity of murine leukemia viruses has often been evaluated for newborn mice, we infected newborn NMRI
of FIS-2 and compared it with that of F-MuL'V clone 57. The result is shown in Fig. 1B. We found that altho)ugh FIS-2 has a restriction map very similar to that of F-MuL,V clone 57, the genomic size of FIS-2 is smaller than that of F--MuLV clone 57 because of the deletion located at the U3 regiion of the LTR. Two extra KpnI sites were found, one locatLed at the LTR region and another close to the EcoRI site. I )ifferent restriction fragment polymorphism between FIS-22 and F-MuLV clone 57 was also observed by digestion wit]h PstI and with SacII. The restriction map of FIS-2 suggested t]hat it is a variant of F-MuLV. Immunosuppressive activity and disease iniduction of FIS-2 in adult and newborn mice. Since FIS-2 i!s a replicationcompetent virus and is structurally related to ,F-MuLV clone 57, we wished to compare its immunosuppres ,sive activity and disease induction with those of F-MuLV clonie 57. The effect of viral infection on the primatry antibody response of the infected adult mice against SRB(C was previously used as a parameter of the immunosuppressiv'e activity of the FV (3, 37) and the primary stock of the FIS vairiant (15). Both the FIS-2 and the F-MuLV clone 57 viruses wi ere injected into adult mice. The mice were immunized with SIRBC at different times after infection, and the numbers of B cells producing antibody against SRBC per 105 splenocytes vovere counted as plaques in a modified Cunningham's assay aftcer immunization for 4 days. The percentage of the number of I3-cell plaques of
TABLE 1. Latency periods and disease specificities induced by FIS-2 and F-MuLV clone 57 in adult and newborn NMRI mice Mouse group and
inocusegrumn
Newborn mice FIS-2 F-MuLV clone 57 Adult mice FIS-2a F-MuLV clone 57
No. of diseased mice/
no. of inoculated inoculate n
Mean latency (days)
(dn atnys
Diagnosis (no. of mice)
No. not
autopsied auopsied
Erythroleukemia
Lymphoma
22/22 18/18
135 1
22 17
1
46
31/31 12/12
333 585
4 3
13 3
l9b
a Biological clone of FIS-2. b Two mice had erythroleukemia and renal disease, 11 mice had renal disease, and 4 mice had erythroleukemia. C All mice had lymphoma, and two mice also had erythroleukemia. d Pneumonia. e Three mice had erythroleukemia. f Unspecified neoplastic disease included tumors in other unspecified organs, such as organs in the digestive system.
5e
Renal disease
Other
13C
id
4f
6980
J. VIROL.
DAI ET AL.
(A)
tn
'a v)
aei
43
Days from inoculation to advanced disease
(B) 100 T
F-MuLV
clone
57
80 + aL)
60 +
x:> co
-FIS-2
40
20 + 0
particular virus and its enhancer element located in the U3 region of the LTR (4, 14, 20), we sequenced the LTR region of FIS-2 and compared it with that of F-MuLV clone 57 (Fig. 4). The result showed a high degree of homology between the LTRs of FIS-2 and F-MuLV clone 57, except that the second copy of the direct repeat of 74 bp had been completely deleted from the FIS-2 LTR. This result was consistent with the deletion observed by restriction mapping. Several nuclear DNA binding factors have been shown to bind to the direct repeats of F-MuLV clone 57 (34). We aligned the sequence of the FIS-2 LTR with that of the F-MuLV LTR and searched for mutations that occurred in these binding sites (Fig. 4). Interestingly, the sequence of the enhancer core element of F-MuLV clone 57, TGTGGTAA, is changed to TGTGGTGA in FIS-2. A single-nucleotide transition of G to A also occurred in the binding site for factor FVa. A methylation interference study indicated that such a mutation affected the binding of the factor (34). Because of the deletion of the second copy of the direct repeat, the binding site for nuclear factor FVbl was missing, and a binding site for the glucocorticoid receptor (GRE), AGAACAGATGG (10, 36), was generated (Fig. 4). We also found that several point mutations occurred in the R region. The U5 region in the FIS-2 LTR is unchanged. Finally, nucleotide sequence analysis confirmed the restriction enzyme sites located in the LTR region as shown by restriction mapping. The loss of PstI and the appearance of an extra KpnI site are caused by substitution of a single nucleotide. Comparison of the nucleotide sequences of the LTRs of FIS-2 and of SFFVp showed that one direct repeat is completely missing in both viruses. Three single nucleotides in the FIS-2 LTR which are different from the F-MuLV sequence are found to be identical to the SFFV sequence at the corresponding positions. One is the substitution of T with C, which causes the loss of the PstI site in the U3 region. The other two are located in the R region (Fig. 4). Since FIS-2 was initially isolated from T helper cells of mice infected with FV, which contains the replication-competent helper virus F-MuLV and the replication defect SFFV, it was possible that FIS-2 was generated by recombination between SFFV and F-MuLV. Predicted amino acid sequence of the FIS-2 gag- and envencoded proteins. To understand the genomic structure of FIS-2 in more detail, we also sequenced the gag and env regions. The amino acid sequences of both genes were deduced and aligned with that of F-MuLV clone 57. A homology of 96% between the two viruses was found for both genes. A total of 38 point mutations occurred scattered over the FIS-2 gag gene, and 24 mutations led to amino acid changes in the gag products (Fig. 5). Among 539 aligned amino acids, 20 were mismatched. Totals of 8, 3, 7, and 2 mutations were located in the MA region, in p12, in CA, and in NC, respectively. Deletion of 3 bp in the MA region caused a loss of one of the three proline residues in the MA protein. Interestingly, this deletion has also been found in two other F-MuLV-related isolates, FB29 and PVC-211 (41, 44). While FB29 induces erythroleukemia, PVC-211 induces neurodegenerative disease. By computer analyses of the Gag protein, we found two extra potential N glycosylation sites (motif Asn-X-Ser/Thr) located in the CA region because of two point mutations. New potential N glycosylation sites have not appeared in FB29 Gag protein but have been found in PVC-211 Gag protein. Since the leader sequence starting from the CUG initiation codon which is used for the synthesis of the gag-encoded cell surface glycoprotein (43) is intact in FIS-2 (data not shown), we assume that the FIS-2 gag-encoded glycoprotein is presented in the infected cells. One of the mutations in the NC region
,,Iv~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 40 Days
80 from
120
inoculation
160 to
200
advanced
240
disease
FIG. 3. Incidence of disease induction in adult (A) and newborn (B) NMRI mice by FIS-2 and F-MuLV clone 57. Adult NMRI mice were infected by biologically cloned FIS-2; all newborn mice were infected by molecularly cloned FIS-2. The differences between the mice infected by FIS-2 and by F-MuLV clone 57 were highly significant in both panel A (P < 0.0003) and panel B (P < 0.0001), as shown by the log rank test.
mice with FIS-2 and F-MuLV clone 57. The diseases induced were studied and compared. Both viruses induced tumors in all mice inoculated as newborns (Table 1). In both groups, the mice developed anemia and gross spleen and often liver enlargement. However, the latency period of disease induction of F-MuLV clone 57 was 80 to 90 days shorter than that of FIS-2 (Fig. 3B). Among a total of 22 infected mice, only one FIS-2-infected mouse showed massive enlargement of lymph nodes consistent with the development of lymphoma; the rest of the infected mice had slightly enlarged lymph nodes. Although microscopic investigations of these slightly enlarged lymph nodes were not performed, macroscopic inspection showed changes very similar to those in the lymph nodes of adult mice with follicle hyperplasia. Kidney enlargement was not observed for either of the groups. These results show that FIS-2 is a relatively weakly leukemogenic variant of F-MuLV with a slightly different disease specificity. The results also indicated that the weaker immunosuppressive activity and the pathogenicity of F-MuLV clone 57 in adult NMRI mice were not due to the weak susceptibility of NMRI mice, since F-MuLV clone 57 could induce erythroleukemia in newborn NMRI mice more rapidly than FIS-2. Nucleotide sequence analysis of the FIS-2 LTR. The differences between FIS-2 and F-MuLV clone 57 shown in their biological properties and by restriction mapping prompted us to analyze the nucleotide sequence of FIS-2. Since there is a direct linkage between the nature of the disease induced by a
by the two viruses
FIS-2 VARIANT OF F-MuLV
VOL. 68, 1994
6981
Pst I
1.F-MuLV 2.FIS-2
rU U3 ATGAAAGACCCCACCAAGTTGCTTAGCCTGATAGCTGCAGTAACGCCATTTTGCAAGG .c. ...................... *
l.F-MuLV CATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGTCAGGTACACGAAA 2.FIS-2.........-.c..-C----oFt LVb Kpn I -j CORE FVa
l.F-MuLV 2.FIS-2
ACAGCT + CGTTGGGCCAAACAGGATATCTGTGGTAAGCAGTTTCGGCCCCGGCCCGG .........A.........................G -.---------FVb2
I
FVb1 DR
1.F-MuLV 2 .F IS -2
GGCCAAGAACAGATACGCTGGGCCAAACAGGATATCTGTGGTAAGCAGTTTCGGCCCG
1.F-MuLV
GTCGGCCCCGGCCCGAGGCCAAGAACGGAT GTCCCCAGATATGGCCCAACCCTCAGC
2 . F IS -2
-
FVb1
l.F-MuLV 2.FIS-2
GRE
AGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTG ..........................................................
CCAAT
l.F-MuLV TGCCTTATTTGAATTAACCAATCAGCCCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCT 2.FIS-2 .............----------T. .--------------------TATA
1.F-MuLV 2.FIS-2
l
~~~~~~~~- R
TCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAG
.........-.c.--------------------C-Kpn I
1.F-MuLV 2.FIS-2
ACTGAGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTGCATCCGACTC T *------....----------G-AT--------.T-.-----------------
1.F-MuLV
GTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCAGAGTGATTGACTACCCGTCTCGGGG
2.FIS-2
1. F-MuLV 2. FIS-2
r-U5
..........................................................
GTCTTTCATT
FIG. 4. Nucleotide sequence of the FIS-2 LTR (line 2) compared with the sequence of the F-MuLV clone 57 LTR (line 1). Homologous sequences are indicated by dots. The direct repeat region (DR) is boxed. The beginnings of the U3, R, and U5 regions are indicated by horizontal arrows. Dashed lines indicate deletions. The binding sites for the previously identified nuclear factors (FVa, FVbl, FVb2, FVc, NF-1, and LVb) and core binding proteins in the F-MuLV clone 57 LTR are overlined. The GRE in the FIS-2 LTR is underlined. The restriction enzyme sites are marked by vertical arrows. The stars indicate the single nucleotide differences in the FIS-2 LTR region that are identical to the SFFV sequence at the corresponding positions.
results in one amino acid substitution in a region that belongs to the conserved motif of three cysteines and one histidine in the retrovirus nucleocapsid protein, which is designated the Cys-His box (35). Comparison between the predicted amino acid sequences of the envelope proteins of FIS-2 and of F-MuLV clone 57 showed that among 676 amino acids, there are 23 amino acid mismatches in total. With the exception of one mismatch of an extra glutamine residue located at the carboxyl-terminal end of the FIS-2 TM protein, all other 22 amino acid changes are distributed over SU region (Fig. 6). Among these 22 amino acid changes, 10 accumulated in a region of 100 amino acids (200 to 300). In particular, in a very short region (282 to 290) there are 3 amino acid changes. Computer analysis showed that such changes gave rise to higher hydrophilicity in the region (282 to 290), which overlaps with the proline-rich
region. Our conclusion is, thus, that although the genomic structure of FIS-2 is highly homologous to that of F-MuLV clone 57, these single point mutations cause amino acid changes in the viral proteins and that some of these changes might influence the structure of the particular protein and further affect its biological properties. DISCUSSION In this report, we describe the molecular cloning and characterization of a variant of F-MuLV, FIS-2. Although the
genomic structure of FIS-2 is very similar to that of F-MuLV clone 57, it displays different pathological phenotypes, including disease specificities and latency period of disease induction following inoculation into both newborn and adult NMRI mice. Many extensive studies have demonstrated that the sequences encompassing the transcriptional enhancer element in the U3 region of the LTR are important determinants for murine leukemia viruses of oncogenicity, disease specificity, and latency period of disease induction (4, 11, 12, 24, 27, 29). We observed that upon inoculation of newborn NMRI mice, FIS-2 induced disease with a longer latency period than that for F-MuLV. This property of FIS-2 is most likely due to the presence of only one direct repeat in the U3 region of FIS-2. This result is consistent with previous studies of the F-MuLV with a deletion of one direct repeat (32). However, the single mutations occurring in some of the binding sites of the transcriptional factors seemed to have a subtle influence on pathogenic differences, including disease specificity, since the lymph nodes in both newborn and adult NMRI mice were obviously more affected by infection with FIS-2 than by infection with F-MuLV clone 57. However, a more careful study of the direct effect of these mutations on the cell-typespecific transcription of the FIS-2 LTR and on the disease specificity of FIS-2 is necessary to establish final conclusions. In contrast to the fact that FIS-2 has a weaker leukemogenicity than F-MuLV in newborn mice, it is more immunosuppressive and pathogenic than F-MuLV when inoculated into
6982
2
1
2
J. VIROL.
DAI ET AL. s sig-pept ide
r>MA
CCA
GM
GCA
60
1
MACSTLSKSPKDKIDPRDLLIPLILFLSLKGARSAAPGSSPHQVYUITWEVTNGDREAVW
60
MGQTVTTPLSLTLDHWKDVERTAHuQSVEIRKRRWVTLCSAEWPTFNVGWPRDGTFNPDI
60
2
MACSTLPKSPKDKIDPRDLLIPLILFLSLKGARSAAPGSSPHQVYEITWEVTNGDRETVW CCA ~ ~~ ~ ~ ~ ~ ~ ~~~C
60
1
AISGNHPLWTWWPDLTPDLCMLALSGPPHWGLEYQAPYSSPPGPPCCSGSSGNSAGCSRD
120
2
AI SGNHPLWTWWPVLTPDLCMLALSGPPHWGLEYQAPYS S PPGPPCCSGS SGS SAGC SRD GTC ~~~~~~~AGC
120
AMCG 1
cc A ITQVKIKVFSPGPHGHPEQVPYIVTWEALAVDPPPWVKPFVHPKP-PLLLPPSAPSLPPE
119
2
ITQVKIKVFSSGPHGHPDQVPYIVTWEALAADPPPWVKPFVHPKPPPLLLPPSAPSLPPE
120
CTCTCA
Fe
p12
PPLSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSSDGNGNS
179
1
2
PPFPTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPSSSDGNGGS TTCCCGTC
180
2
1GCP=GPLG 2 GEAAPTEGAPDSSPMVSRLRGRREPPVADSTTSQAFPLRLGGNGQLQYWPFSSSDLYNWK
239
2
GEVAPTEGAPDS SPMVSRLRGRREP PVADSTTSQAFPLRQGGNGQFQYWPFS SSDLYNWK
24 0
1
NNPSFSEDPGKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDG
299
2
NNNPSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDG
300
1
RPTQLPNEINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQ
359
2
RPTQLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQ
360
1
GPL*SPSAFLERLKEAYRRYTPYDPEDPGQETCVSMSFIWQSAPDIGRKLERLEGLGWT
419
2
GPkIESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKT AGC
420
1
LGDLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDEQREKERDRRRHREMSKLLA
479
2
LGDLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDEQREKERDRRRHREMSKLLA
480
1
TVISGQRQDRQGGERRRPQLDHDQCAYCKEKGHWAKDCPKKPRGPRGPRPQASLLTLDDO
539
TVISGQRQDRQGGERRRPQLDHDCYKKHADPKKPRGPRGPRPQASLLTLDDO
540
[7
A
AK
CNEPLTSLTPRCNTAWNRLKLDQVTHKSSEGFYVCPGSHRPREAKSCGGPDSFYCASWGC
180
CDEPLTSLTPRCNTAWNRLKLDQVTHKSSEGFYVCPGSHRPREAKSCGGPDSFYCASWGC
180
G7AC
AGA
AAC
[CA
TVVSGRQDRGGERRPLDHCA
AAC
GAC
1
2
F> su
MGQTVTTPLSLTLDHWKDVERTAHMQSVEVRKRRWVTLCSAEWPTFNVGWPQDGTFNPDI
ETTGRVYWKPSSSWDYITVDNALTTNQAVQVCKDNKWCNPLAIRFTNAGRQVTSWTTGHS
240
2
ETTGRVYWKPSSSWDYITVDNNILTTSQAVQVCKDNKWCNPLAIQFTNAGKQVTSWTTGHY
240
1
WGLRLYVTGKDPGLTFGIRLKYQNLGPRVPIGPNPVLADQLSFP,LEPNPOKPAKSPPASkl
300
2
WGLRLYVSGRDPGLTFGIRLRYQNLGPRVPIGPNPVLADQLSLPRPNPLPKPAKSPPASII AGA C=C CA -CT = G
300
A
c
AU
A
AAC C
1
STPTLISPSPTPTQPPPAGTGDRLLNLVQGAYQALuLTNPDKTQECWLCLVSGPPYYEGV
360
2
STPTLISPSPTPTQPPPAGTGDRLLNLVQGAYQAL,.LTNPDKTQECWLCLVSGPPYYEGV
360
1
AGAGGC GCGQ AVLGTYSwHTSAPAaCSAASQHKLTLSEVTGRGLCIGTVPKTHQALCrTTLKTGKGSYYL
420
2
AVLGTYS0HTSAPAUCSVASQHKLTLSEVTGRGLCIGTVPKTHQALCNTTLKIDKGSYYL
420
GTG
ATAGAC
AACFrm
NC
AA
TCT
1
VAPAGTMWACNTGLTPCLSATVI4HRTTDYCVLVELWPRVTYHPPSYVYSQFENSYRHKRE
480
2
VAPTGTIWACNTGLTPCLSATVLaRTTDYCVLVELWPRVTYHPPSYVYSQFEKSYRHKRE AA ACA AM
480
1
PVSLTLALLLGGLTMGGIAAGVGTGTTALVATQQFQQLHAAVQDDLKEVEKSITNLEKSL
540
2
PVSLTLALLLGGLTMGGIAAGVGTGTTALVATQQFQQLHAAVQDDLKEVEKSITNLEKSL
540
2
TSLSEVVLQH8aLDLLFLKE=2CAALKEECCFYADHTGLVRDSMAKLRERLTQRQKLF
600
1
ESSQGWFEGLFNRSPWFTTLISTIMGPLIILLLILLFGPCILNRLVQFVKDRISVVQALV
660
2
ESSQGWFEGLFNRSPWFTTLISTIMGPLIILLLILLFGPCILNRLVQFVKDRISSWQALV
660
AGA
FIG. 5. Comparison between the predicted amino acid sequences of the gag-encoded proteins of FIS-2 (line 1) and of F-MuLV clone 57 (line 2). The nucleotide change that leads to amino acid substitution is shown above the corresponding amino acid. The potential N glycosylation sites in the Gag proteins of both viruses are underlined, and two extra glycosylation sites are marked by asterisks. The MA, p12, CA, and NC regions are indicated by horizontal arrows. The loss of one proline residue in the MA region of FIS-2 is marked by a dash. The sequence of the Cys-His box is underlined.
immunocompetent, adult mice. We showed that FIS-2 is able to induce profound suppression of the primary antibody response rapidly and persistently. Although the relationship between retrovirus-induced immunosuppression and leukemogenicity is still unclear, the renal disease induced by FIS-2 in adult mice, which is caused by deposition of immune complex in the renal glomeruli, has been considered a consequence of immune dysfunction. Hence, our data demonstrated that FIS-2 is a weakly leukemogenic, relatively strongly immunosuppressive variant of F-MuLV. It is known that the transmembrane envelope protein TM has a direct immunosuppressive effect (8, 47) and is capable of inhibiting the proliferative response of lymphocytes to mitogen by blocking the production of interleukin-2 in lymphocytes (9, 21, 39, 46). This inhibitory activity has been mapped to a specific region containing 17 amino acid residues in TM, which belongs to a highly conserved region of TM among the retroviruses of all species (7). In the present study, we showed that in FIS-2 as well as in F-MuLV clone 57, this region remains unchanged (Fig. 6). Also, the predicted amino acid sequence of FIS-2 TM is almost identical with that of F-MuLV clone 57. Therefore, regions other than TM in the FIS-2
600
c.M 1
LTQQYHQLKPLEYEPQO
677
2
LTQQYHQLKPLEYEP-O
676
FIG. 6. Comparison of the predicted amino acid sequences of the env-encoded proteins of FIS-2 (line 1) and of F-MuLV clone 57 (line 2). The regions of the signal peptide (sig-peptide), SU, and TM are indicated. The region (282 to 290) with a cluster of mutations in the FIS-2 SU region is underlined. In addition, the region (548 to 564) containing 17 amino acids which was shown to have immunosuppressive activity in TM is underlined (see Discussion). The nucleotide change that leads to amino acid substitution is shown above the corresponding amino acid.
genome whose sequences are different from that of F-MuLV clone 57 might be responsible for the generation of the difference in immunosuppressive activities between the two viruses. In other words, there exists another region(s) in the FIS-2 genome that can affect the immune function either directly or in conjunction with TM. The best example of immunosuppressive activity of a retroviral protein other than TM comes from the studies of the defective murine AIDS virus MAIDS. The determinant for the pathogenicity of this virus was located in the MA and p12 regions of the gag gene (42). Interestingly, the gag-encoded surface protein of MAIDS has the ability to stimulate a proliferative response of normal spleen cells in vitro by expansion of a population of T cells with specific TCR, VB5, and
VOL. 68, 1994
FIS-2 VARIANT OF F-MuLV
VBll and thus exhibits many characteristics compatible with the definition of a superantigen (26). Recently, it was found that the Gag protein Pr55 of human immunodeficiency virus type 1 binds to cellular protein cyclophilins A and B, which are the cellular receptors for the immunosuppressive drug cyclosporin A, and that this binding has been suggested to play an important role in the pathogenesis of AIDS (33). In addition, the envelope glycoprotein of murine leukemia virus was reported to have a mitogenic effect by binding to the cellular receptors of growth factors (30, 31, 49). Nucleotide sequence analyses showed several interesting mutations in the structural genes of FIS-2 despite its high homology with F-MuLV clone 57. It is particularly notable that both the gag- and the env-encoded viral glycoproteins which are found on the surfaces of infected cells are affected by the mutations. Since FIS-2 is able to induce lymphoid hyperplasia to a great extent, it would be interesting to investigate the mitogenic effects of the FIS-2 viral proteins. Previous studies showed that F-MuLV-specific cytotoxic lymphocytes are able to lyse the infected cells by recognition of cell surface proteins encoded by F-MuLV env and gag in the context of major histocompatibility complex class I molecules (25). Previous studies also demonstrated that protein glycosylation can influence antigen recognition by antibody and by cytotoxic T lymphocytes (2). It is possible that mutations in FIS-2 gag and env genes have caused the loss of antigenic epitopes and enable it to evade the immune response against F-MuLV and F-MuLV-infected cells. The phenomenon of the loss of epitopes by generation of variants has been described for lentiviruses such as simian immunodeficiency virus (40) and human immunodeficiency virus (2). Recently, it has also been demonstrated for murine retrovirus (50). In summary, we have molecularly cloned and characterized a replication-competent F-MuLV-related but weakly leukemogenic and relatively strongly immunosuppressive virus, FIS-2. We are currently concentrating on studies of the mutations observed in the FIS-2 genome and their roles in induction of
immunosuppression. We believe that FIS-2 represents another interesting murine model for studying the dysfunction of immune-competent cells caused by retrovirus infections. ACKNOWLEDGMENTS We thank Lars Vatten for help with statistical analyses. We also thank Finn Skou Pedersen and Hans Krokan for careful reading of the
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