VIROLOGY
64,49-62
(197%
A Replication which ROBERT
Defective
Mutant
Fails to Make a Functional
R. FRIIS,*
Department
WILLIAM
of Microbiology,
of Rous Sarcoma
Reverse Transcriptase’
S. MASON,3 YOUNG HALPERN’ School of Medicine, University Los Angeles, California 90033 Accepted
Virus
C. CHEN,
of Southern
AND
MICHAEL
S.
California,
October 18, 1974
ts 672 is a replication defective temperature sensitive mutant of Rous sarcoma virus which produces large yields of noninfectious viral particles (NI 672) during growth in cells at the nonpermissive temperature. This study has been directed to identifying the temperature sensitive function of ts 672 by analysis of the NI 672 particles and by comparing this mutation to other known mutations in functions affecting replication. The only structural defect observed with NI 672 was the absence of the virion-associated RNA dependent DNA polymerase activity characteristic of all infectious RNA tumor viruses. Although there are other viral mutants which exhibit an altered polymerase activity, ts 672 is unique in that the DNA polymerase is only temperature sensitive prior to or during the assembly of the virus particle. The DNA polymerase activity of the ts 672 virions produced at the permissive temperature is just as temperature stable as the wild type enzyme. Mixed infection tests performed with ts 672 and with two other DNA polymerase mutants have failed to show a complementation, thus supporting the idea that ts 672 is defective in the DNA polymerase function. Recombination studies with ts 672 and an avian leukosis virus have shown that there is a very high degree of genetic linkage between the temperature sensitive DNA polymerase function of ts 672 and the determinants for the host range of these viruses, the viral envelope proteins.
possesses a mutation in a function which is required for replication of infectious progThe characteristics of a replication deeny, but not for the transformation of fective temperature sensitive mutant of infected cells and (2) it is able to produce Rous sarcoma virus have been described (Friis and Hunter, 1973). This virus, ts 672, large numbers of noninfectious viral partiis unique among the known avian sarcoma cles when infected cells are maintained at virus mutant isolates in two respects: (1) it the nonpermissive temperature. The experiments to be described in this communication were designed to reveal the ‘This study was supported by Contract No. NOlnature of the specific lesion(s) which ts 672 CP-43242 from the National Cancer Institute and by imposes on its progeny produced at the Grant No. AI-lo, 809.01 from the National Institute of nonpermissive temperature. Since noninAllergy and Infectious Diseases. fectious viral particles, hereafter to be 2Pre~ent address: Institut fur Virologie, Justus referred to as NI 672, were available for Liebig-Universitat, 6300 Giessen, Frankfurter Strasse biological and biochemical analysis, we 107, Germany. first determined which components were $Fellow of the Leukemia Society of America. Present address: Institute for Cancer Research, 7701 altered or lacking. Then, having obtained a preliminary conclusion about the nature of Burholme Avenue, Philadelphia. PA 19111. the defect, ts 672 was compared with other “Present address: Wistar Institute, Thirty-sixth Street at Spruce, Philadelphia, PA 19104. avian sarcoma virus mutants using in viva INTRODUCTION
50
FRIIS ET AL.
complementation tests. Finally, the genetic linkage of the temperature sensitive marker to the marker for viral host range was measured in recombination tests with an avian leukosis virus. MATERIAL
AND METHODS
Viruses and Cells ts LA 672 is a temperature
infectious BH RSV( -) in the absence of a helper virus, they may be very conveniently used to assay helper viruses of subgroups A, C, and E, to which quail cells are susceptible. One infectious unit is sufficient over the course of 2-3 days to result in a large yield of infectious BH RSV pseudotype. A chf test based on this principle has been devised, using instead of helper avian leukosis viruses, the chick embryo cells under test. When 2 x lo5 chf positive chick embryo cells are cocultivated with an equal number of BH RSV( -)Q for 3 days, a yield of over lo3 FFU/ml BH RSV pseudotype is obtained. If chf negative chick embryo cells are employed, no infectious virus is observed. This test for chf depends on cell fusion between chick and quail cells which occurs spontaneously at a rate of about at least one per 10’ cells per day. This test works well regardless of the susceptibility phenotype for avian tumor viruses of the chicken cells employed, and the sensitivity of the method is equivalent to the procedure of Weiss et al. (1973). Virus stocks were cloned by isolation of single foci from monolayers of chf negative cells. All experiments performed during the course of studies with ts 672 were done using chf negative cells. For these studies either Hamm F10 or Dulbecco’s modified Eagles medium were used. These media were always supplemented with 5% calf serum and 10% tryptose phosphate broth. Focus tests were performed essentially as described by Rubin (1960), except that during the virus adsorption period Polybrene (Aldrich Chemical Co.) at a final concentration of 2 pglml was included in the medium (Toyoshima and Vogt, 1968). Dimethyl sulfoxide (1%) was added to medium for preparation of virus stocks, both because of enhanced cell monolayer stability and increased virus yield (Vogt et al., 1970).
sensitive mutant of the Prague strain of Rous sarcoma virus, subgroup A (wt PR-A) and was isolated as previously described (Friis and Hunter, 1973). The permissive temperature for its growth is 35”, and the nonpermissive temperature, 41’. The Bryan high titer strain of Rous sarcoma virus, cloned free of helper viruses (BH RSV(-)), was employed in these experiments as a model for mutants defective in a function required for the establishment of virus infection (Hanafusa et al., 1964). An avian leukosis virus, Rous associated virus type 6, subgroup B (RAV-6), was used in recombination experiments with ts LA 335 and ts LA 337, two temperature sensitive mutants of the Prague strain of Rous sarcoma virus, subgroup C (wt PR-C) which have been shown to contain a temperature sensitive RNA dependent DNA polymerase (Linial and Mason, 1973; Mason et al., 1974), were employed in complementation tests with ts 672. Chick embryos from avian leukosis virus-free flocks were generously made available by Mr. Richard Raymond, Heisdorf and Nelson Farms, Redmond, WA, and by Dr. E. Vielitz, Lohmann Tierzucht GmbH, Cuxhaven, Germany. These cells were tested for the presence of the chick helper factor (chf) (Hanafusa et al., 1970) according to the procedure described by Weiss et al. (1973). In addition, a second method for detection of chf was employed in testing most cells; this procedure will be briefly described. A Japanese quail cell line chronically infected with BH RSV(-) was initiated in our laboratory in 1971 (Friis, 1972). Virus Penetration Using Sendai Virus Mediated Cell Fusion This cell line has been designated BH RSV( -) Q clone 3 and has been in continuPara-influenza virus type I strain Senous culture for over two years and some 120 dail52 from the American Type Culture passages. Since the cells produce only non- Collection was prepared in the allantoic
cavity of lo- to 11-day-old embryonated eggs. After 3 days incubation, allantoic fluids were harvested which could be shown to contain 2000-4000 hemagglutinating units (HAU)/ml. Virus was pelleted from the fluids by centrifugation by treatment and was inactivated with 1:4000-diluted beta-propiolactone (Matheson, Coleman and Bell). After inactivation, the hemagglutination titer was found to be 2000-4000 HAU/ml. The inactivated virus suspension was stored at -70” C in 1 ml lots for subsequent use in fusion experiments. Inactivated Sendai virus was used to facilitate the penetration of cells by viruses which were normally unable to penetrate because of defective viral envelope proteins or the absence of receptors for virus at the cell surface. The virus under test was mixed with a cell suspension containing 1 x lo7 chick embryo cells to a volume of 1 ml, to which was added 0.2 ml of inactivated Sendai virus (2000-4000 HAU/ml). The mixture were incubated for 30 min at O”, followed by a 20 min incubation at 37”. After incubation, 5 ml of medium was added, and the suspended cells were pelleted by centrifugation at 600 g for 5 min. The cell pellet was gently resuspended into warm medium and the cells were plated at different dilutions in 60 mm Falcon plastic culture dishes for an infectious center assay at 35”. Radioisotopic Labeling, Purification, and Physical Characterization of Viral Components For comparative studies of NI 672 and ts 672 proteins and RNA components, radioisotopic labeling was performed with the appropriate precursors as previously described (Vogt and Friis, 1971; Weiss et al., 1971; Halpern et al., 1973). In particular, proteins prepared for coelectrophoresis had been labeled by synthetic amino acid mixtures containing either 14C- or 3H-amino acids in the same relative proportions (New England Nuclear Corp., Boston, MA). Virus purification from tissue culture supernatants was accomplished according
to the methods described by Halpern et al. (1973). Viral structural proteins were analyzed using discontinuous SDS polyacrylamide gels according to the technique of Laemmli (1969) as employed by Halpern et al. (1973). RNA was extracted from virus as described by Duesberg (1968), and the methods for analysis using velocity sedimentation on gradients of lo-30% glycerol were the same as reported by Halpern et al. (1973). Assays for RNA Polymerase
Dependent
DNA
Virus that had been pelleted from several liters of tissue culture supernatant was gradient purified and the protein concentration of the resulting virus suspension determined by the method of Lowry et al. (1951) using bovine serum albumin as a calibration standard. The conditions for the DNA polymerase assay were exactly as described previously (Linial and Mason, 1973). The precipitation of the reaction products were performed as reported by Mason et al. (1974). Technique for the Selection nant Viruses
of Recombi-
It has been demonstrated that RNA tumor viruses may recombine genetic markers among the progeny of a mixed infection (Vogt, 1971; Kawai and Hanafusa, 1972). Since ts 672 is defective in a replication function of the virus, it is possible to complement this function with an avian leukosis virus (Friis and Hunter, 1973), and it should also be possible to recombine this defective sarcoma virus with an avian leukosis virus to obtain a wild type sarcoma virus. In the course of doing the latter experiment, and for reasons of expedience in selecting for recombinants, the host range marker of the respective viruses was also examined. Mixed infection was established with ts 672, subgroup A, and an avian leukosis virus, RAV-6, subgroup B, at 35”. When transformation indicated confluent infection with ts 672, supernatant samples were harvested as
52
FRIIS ET AL.
stocks. The plan was to select for and test only recombinants, taking advantage of the fact that all viruses that were genotypitally sarcoma viruses, i.e., focus-forming viruses in tissue culture, and which were members of subgroup B on the basis of the host range marker must have been recombinants. In order to observe directly the genotypic character of the virus it was necessary to inoculate the stock from mixed infection first onto susceptible cells at a dilution high enough so that simultaneous infection of a cell by two viruses was highly improbable, then harvesting the virus progeny after only 30 hr, before a second cycle could lead again to phenotypic mixing. This second stock was one, then, in which the host range determinant on the viral surface was determined by its own genome. This stock was then selected against the ts 672 parental type by application of antiserum specific against subgroup A. A focus test was performed, and after 8 days, single, widely separated foci were aspirated. These presumptive recombinants were then subjected to two further stages of single focus cloning. In order to ascertain whether these isolates were free of contamination with the original RAV-6 parent after this cloning procedure, a combined focus test-plaque test was performed. The plaque test procedure reported by Graf (1972) was employed. By marking the site of each focus appearing after 8 days incubation, and comparing these with the locations of plaques obtained with the aid of neutral red staining after 13 days incubation, it was possible to conclude that the cloned isolates were free of contaminating leukosis virus, i.e, a virus that formed plaques but failed to form foci. By this procedure, a collection of virus clones was obtained which were recombinant with RAV-6 as evidenced by the presence of the genetic determinant for the subgroup B viral envelope antigen. The inheritance from the ts 672 parent was obvious from the fact that all of these clones were sarcoma viruses, hence exhibiting a genetic marker of ts 672 which could not be observed with RAV-6. The temperature sensitivity of these clones was then individually assayed.
RESULTS
The
Defect is Not Penetration
at
the
Level
of
The Bryan high titer strain of Rous sarcoma virus (BH RSV) exhibits some properties which are similar to those of ts 672. In particular, BH RSV produces large numbers of noninfectious progeny when infection is established in cells lacking a helper virus or the chick helper factor (chf) (Hanafusa et al., 1964; Hanafusa et al., 1970). These progeny are noninfectious because they lack a functional viral envelope protein (Hanafusa et al. 1964; Scheele and Hanafusa, 1972), which is essential for penetration of the virus into the host cells. In mixed infection, BH RSV always takes on the viral envelope properties of the helper virus. ts 672 is also readily complemented by avian leukosis viruses; however, it is noteworthy that ts 672 does appear to produce sufficient viral envelope antigen of subgroup A at 41” to produce interference against potential subgroup A helper viruses, since complementation of ts 672 at the nonpermissive temperature was observed following superinfection with all helper avian leukosis viruses tested except those belonging to subgroup A (Friis and Hunter, 1973). In order to determine whether the defect in ts 672 is like that of BH RSV, a procedure was adopted which allows the introduction of the virus into the cell without the usual dependence on viral envelope-host receptor interactions. This technique is the fusion of virus particles with the host cell as a result of treatment with the Sendai virus fusion factor. The experiment was performed with NI 672 using BH RSV as a known penetration defective control. The results are summarized in Table 1. These data show that when NI 672 was introduced into the cell with fusion factor, no significant rise in the rate of successful infection over the background of “leaky” virus present in all NI 672 stocks was observed. For this experiment, it must be noted that C/A phenotype chick embryo cells were employed and that these cells are
A REPLICATION TABLE
DEFECTIVE
1
MUTANT
OF RSV
53
almost totally resistant to subgroup A viruses including ts 672. This was done to assure that the penetration observed after fusion factor treatment was independent of viral envelope-host receptor interaction. In this way, it was possible to exclude the background of “leaky” virus that is always present at titers of lo3 to 10’ FFU/ml in NI 672 preparations. Controls are shown in which the infections were performed with C/BDE phenotype cells which are fully susceptible to ts 672 and to the “leaky” virus present in NI 672. This control in fact demonstrates the importance of reducing the background through the use of resistant cells.
pears unlikely to demonstrate the particular lesion as a striking change in the rate of migration of a particular protein. The probability is much greater, however, that since a particular defective protein fails to function in its designated role in conferring infectivity, that it will also fail to associate correctly with other components at the time of virus maturation. If such were to be the case, analysis of the proteins of NI 672 compared to ts 672 might be expected to reveal quantitative differences in the amount of a particular protein incorporated into virus. It might be deficient, in excess, or totally absent. For this experiment, “C- and 3H-labeled amino acid mixtures of identical composition and relative activities were used so that a strict comparison of coelectrophoresed viral proteins would be possible. Figure 1 shows the result of the coelectrophoresis which failed to show any difference between NI 672 and ts 672. Similar coelectrophoresis between wild type PR-A grown at 41 C and NI 672 also indicated no aberration in the components of NI 672 (data not shown). Likewise, use of [“Cl- and [3H]glucosamine as precursors for labeling viral glycoproteins failed to indicate a structural defect in NI 672 (data not shown). Although there is always a low level of infectious virus in NI 672 preparations resulting from “leaky” function, this virus represents a fraction of less than 0.1% of the total present, and would not be expected to obscure real structural differences between NI 672 and ts 672. Therefore, while one cannot rule out the possibility of a subtle change in the structure of a protein which destroys its biological function without impairing its assembly into the viral structure, it seems likely that the structural proteins of NI 672 do not differ from those of ts 672.
The Structural
The RNA of NI 672
VIRAL PENETRATION MEDIATED BY SENDAI VIRUS FUSION FACTOR Virus
Cell phenotype
Observed titers (FFU/mlln Sendai treatment
Control
BH RSV( - I
C/BDE CIA
1 x loa 2 x lo3
64,49-62
(197%
A Replication which ROBERT
Defective
Mutant
Fails to Make a Functional
R. FRIIS,*
Department
WILLIAM
of Microbiology,
of Rous Sarcoma
Reverse Transcriptase’
S. MASON,3 YOUNG HALPERN’ School of Medicine, University Los Angeles, California 90033 Accepted
Virus
C. CHEN,
of Southern
AND
MICHAEL
S.
California,
October 18, 1974
ts 672 is a replication defective temperature sensitive mutant of Rous sarcoma virus which produces large yields of noninfectious viral particles (NI 672) during growth in cells at the nonpermissive temperature. This study has been directed to identifying the temperature sensitive function of ts 672 by analysis of the NI 672 particles and by comparing this mutation to other known mutations in functions affecting replication. The only structural defect observed with NI 672 was the absence of the virion-associated RNA dependent DNA polymerase activity characteristic of all infectious RNA tumor viruses. Although there are other viral mutants which exhibit an altered polymerase activity, ts 672 is unique in that the DNA polymerase is only temperature sensitive prior to or during the assembly of the virus particle. The DNA polymerase activity of the ts 672 virions produced at the permissive temperature is just as temperature stable as the wild type enzyme. Mixed infection tests performed with ts 672 and with two other DNA polymerase mutants have failed to show a complementation, thus supporting the idea that ts 672 is defective in the DNA polymerase function. Recombination studies with ts 672 and an avian leukosis virus have shown that there is a very high degree of genetic linkage between the temperature sensitive DNA polymerase function of ts 672 and the determinants for the host range of these viruses, the viral envelope proteins.
possesses a mutation in a function which is required for replication of infectious progThe characteristics of a replication deeny, but not for the transformation of fective temperature sensitive mutant of infected cells and (2) it is able to produce Rous sarcoma virus have been described (Friis and Hunter, 1973). This virus, ts 672, large numbers of noninfectious viral partiis unique among the known avian sarcoma cles when infected cells are maintained at virus mutant isolates in two respects: (1) it the nonpermissive temperature. The experiments to be described in this communication were designed to reveal the ‘This study was supported by Contract No. NOlnature of the specific lesion(s) which ts 672 CP-43242 from the National Cancer Institute and by imposes on its progeny produced at the Grant No. AI-lo, 809.01 from the National Institute of nonpermissive temperature. Since noninAllergy and Infectious Diseases. fectious viral particles, hereafter to be 2Pre~ent address: Institut fur Virologie, Justus referred to as NI 672, were available for Liebig-Universitat, 6300 Giessen, Frankfurter Strasse biological and biochemical analysis, we 107, Germany. first determined which components were $Fellow of the Leukemia Society of America. Present address: Institute for Cancer Research, 7701 altered or lacking. Then, having obtained a preliminary conclusion about the nature of Burholme Avenue, Philadelphia. PA 19111. the defect, ts 672 was compared with other “Present address: Wistar Institute, Thirty-sixth Street at Spruce, Philadelphia, PA 19104. avian sarcoma virus mutants using in viva INTRODUCTION
50
FRIIS ET AL.
complementation tests. Finally, the genetic linkage of the temperature sensitive marker to the marker for viral host range was measured in recombination tests with an avian leukosis virus. MATERIAL
AND METHODS
Viruses and Cells ts LA 672 is a temperature
infectious BH RSV( -) in the absence of a helper virus, they may be very conveniently used to assay helper viruses of subgroups A, C, and E, to which quail cells are susceptible. One infectious unit is sufficient over the course of 2-3 days to result in a large yield of infectious BH RSV pseudotype. A chf test based on this principle has been devised, using instead of helper avian leukosis viruses, the chick embryo cells under test. When 2 x lo5 chf positive chick embryo cells are cocultivated with an equal number of BH RSV( -)Q for 3 days, a yield of over lo3 FFU/ml BH RSV pseudotype is obtained. If chf negative chick embryo cells are employed, no infectious virus is observed. This test for chf depends on cell fusion between chick and quail cells which occurs spontaneously at a rate of about at least one per 10’ cells per day. This test works well regardless of the susceptibility phenotype for avian tumor viruses of the chicken cells employed, and the sensitivity of the method is equivalent to the procedure of Weiss et al. (1973). Virus stocks were cloned by isolation of single foci from monolayers of chf negative cells. All experiments performed during the course of studies with ts 672 were done using chf negative cells. For these studies either Hamm F10 or Dulbecco’s modified Eagles medium were used. These media were always supplemented with 5% calf serum and 10% tryptose phosphate broth. Focus tests were performed essentially as described by Rubin (1960), except that during the virus adsorption period Polybrene (Aldrich Chemical Co.) at a final concentration of 2 pglml was included in the medium (Toyoshima and Vogt, 1968). Dimethyl sulfoxide (1%) was added to medium for preparation of virus stocks, both because of enhanced cell monolayer stability and increased virus yield (Vogt et al., 1970).
sensitive mutant of the Prague strain of Rous sarcoma virus, subgroup A (wt PR-A) and was isolated as previously described (Friis and Hunter, 1973). The permissive temperature for its growth is 35”, and the nonpermissive temperature, 41’. The Bryan high titer strain of Rous sarcoma virus, cloned free of helper viruses (BH RSV(-)), was employed in these experiments as a model for mutants defective in a function required for the establishment of virus infection (Hanafusa et al., 1964). An avian leukosis virus, Rous associated virus type 6, subgroup B (RAV-6), was used in recombination experiments with ts LA 335 and ts LA 337, two temperature sensitive mutants of the Prague strain of Rous sarcoma virus, subgroup C (wt PR-C) which have been shown to contain a temperature sensitive RNA dependent DNA polymerase (Linial and Mason, 1973; Mason et al., 1974), were employed in complementation tests with ts 672. Chick embryos from avian leukosis virus-free flocks were generously made available by Mr. Richard Raymond, Heisdorf and Nelson Farms, Redmond, WA, and by Dr. E. Vielitz, Lohmann Tierzucht GmbH, Cuxhaven, Germany. These cells were tested for the presence of the chick helper factor (chf) (Hanafusa et al., 1970) according to the procedure described by Weiss et al. (1973). In addition, a second method for detection of chf was employed in testing most cells; this procedure will be briefly described. A Japanese quail cell line chronically infected with BH RSV(-) was initiated in our laboratory in 1971 (Friis, 1972). Virus Penetration Using Sendai Virus Mediated Cell Fusion This cell line has been designated BH RSV( -) Q clone 3 and has been in continuPara-influenza virus type I strain Senous culture for over two years and some 120 dail52 from the American Type Culture passages. Since the cells produce only non- Collection was prepared in the allantoic
cavity of lo- to 11-day-old embryonated eggs. After 3 days incubation, allantoic fluids were harvested which could be shown to contain 2000-4000 hemagglutinating units (HAU)/ml. Virus was pelleted from the fluids by centrifugation by treatment and was inactivated with 1:4000-diluted beta-propiolactone (Matheson, Coleman and Bell). After inactivation, the hemagglutination titer was found to be 2000-4000 HAU/ml. The inactivated virus suspension was stored at -70” C in 1 ml lots for subsequent use in fusion experiments. Inactivated Sendai virus was used to facilitate the penetration of cells by viruses which were normally unable to penetrate because of defective viral envelope proteins or the absence of receptors for virus at the cell surface. The virus under test was mixed with a cell suspension containing 1 x lo7 chick embryo cells to a volume of 1 ml, to which was added 0.2 ml of inactivated Sendai virus (2000-4000 HAU/ml). The mixture were incubated for 30 min at O”, followed by a 20 min incubation at 37”. After incubation, 5 ml of medium was added, and the suspended cells were pelleted by centrifugation at 600 g for 5 min. The cell pellet was gently resuspended into warm medium and the cells were plated at different dilutions in 60 mm Falcon plastic culture dishes for an infectious center assay at 35”. Radioisotopic Labeling, Purification, and Physical Characterization of Viral Components For comparative studies of NI 672 and ts 672 proteins and RNA components, radioisotopic labeling was performed with the appropriate precursors as previously described (Vogt and Friis, 1971; Weiss et al., 1971; Halpern et al., 1973). In particular, proteins prepared for coelectrophoresis had been labeled by synthetic amino acid mixtures containing either 14C- or 3H-amino acids in the same relative proportions (New England Nuclear Corp., Boston, MA). Virus purification from tissue culture supernatants was accomplished according
to the methods described by Halpern et al. (1973). Viral structural proteins were analyzed using discontinuous SDS polyacrylamide gels according to the technique of Laemmli (1969) as employed by Halpern et al. (1973). RNA was extracted from virus as described by Duesberg (1968), and the methods for analysis using velocity sedimentation on gradients of lo-30% glycerol were the same as reported by Halpern et al. (1973). Assays for RNA Polymerase
Dependent
DNA
Virus that had been pelleted from several liters of tissue culture supernatant was gradient purified and the protein concentration of the resulting virus suspension determined by the method of Lowry et al. (1951) using bovine serum albumin as a calibration standard. The conditions for the DNA polymerase assay were exactly as described previously (Linial and Mason, 1973). The precipitation of the reaction products were performed as reported by Mason et al. (1974). Technique for the Selection nant Viruses
of Recombi-
It has been demonstrated that RNA tumor viruses may recombine genetic markers among the progeny of a mixed infection (Vogt, 1971; Kawai and Hanafusa, 1972). Since ts 672 is defective in a replication function of the virus, it is possible to complement this function with an avian leukosis virus (Friis and Hunter, 1973), and it should also be possible to recombine this defective sarcoma virus with an avian leukosis virus to obtain a wild type sarcoma virus. In the course of doing the latter experiment, and for reasons of expedience in selecting for recombinants, the host range marker of the respective viruses was also examined. Mixed infection was established with ts 672, subgroup A, and an avian leukosis virus, RAV-6, subgroup B, at 35”. When transformation indicated confluent infection with ts 672, supernatant samples were harvested as
52
FRIIS ET AL.
stocks. The plan was to select for and test only recombinants, taking advantage of the fact that all viruses that were genotypitally sarcoma viruses, i.e., focus-forming viruses in tissue culture, and which were members of subgroup B on the basis of the host range marker must have been recombinants. In order to observe directly the genotypic character of the virus it was necessary to inoculate the stock from mixed infection first onto susceptible cells at a dilution high enough so that simultaneous infection of a cell by two viruses was highly improbable, then harvesting the virus progeny after only 30 hr, before a second cycle could lead again to phenotypic mixing. This second stock was one, then, in which the host range determinant on the viral surface was determined by its own genome. This stock was then selected against the ts 672 parental type by application of antiserum specific against subgroup A. A focus test was performed, and after 8 days, single, widely separated foci were aspirated. These presumptive recombinants were then subjected to two further stages of single focus cloning. In order to ascertain whether these isolates were free of contamination with the original RAV-6 parent after this cloning procedure, a combined focus test-plaque test was performed. The plaque test procedure reported by Graf (1972) was employed. By marking the site of each focus appearing after 8 days incubation, and comparing these with the locations of plaques obtained with the aid of neutral red staining after 13 days incubation, it was possible to conclude that the cloned isolates were free of contaminating leukosis virus, i.e, a virus that formed plaques but failed to form foci. By this procedure, a collection of virus clones was obtained which were recombinant with RAV-6 as evidenced by the presence of the genetic determinant for the subgroup B viral envelope antigen. The inheritance from the ts 672 parent was obvious from the fact that all of these clones were sarcoma viruses, hence exhibiting a genetic marker of ts 672 which could not be observed with RAV-6. The temperature sensitivity of these clones was then individually assayed.
RESULTS
The
Defect is Not Penetration
at
the
Level
of
The Bryan high titer strain of Rous sarcoma virus (BH RSV) exhibits some properties which are similar to those of ts 672. In particular, BH RSV produces large numbers of noninfectious progeny when infection is established in cells lacking a helper virus or the chick helper factor (chf) (Hanafusa et al., 1964; Hanafusa et al., 1970). These progeny are noninfectious because they lack a functional viral envelope protein (Hanafusa et al. 1964; Scheele and Hanafusa, 1972), which is essential for penetration of the virus into the host cells. In mixed infection, BH RSV always takes on the viral envelope properties of the helper virus. ts 672 is also readily complemented by avian leukosis viruses; however, it is noteworthy that ts 672 does appear to produce sufficient viral envelope antigen of subgroup A at 41” to produce interference against potential subgroup A helper viruses, since complementation of ts 672 at the nonpermissive temperature was observed following superinfection with all helper avian leukosis viruses tested except those belonging to subgroup A (Friis and Hunter, 1973). In order to determine whether the defect in ts 672 is like that of BH RSV, a procedure was adopted which allows the introduction of the virus into the cell without the usual dependence on viral envelope-host receptor interactions. This technique is the fusion of virus particles with the host cell as a result of treatment with the Sendai virus fusion factor. The experiment was performed with NI 672 using BH RSV as a known penetration defective control. The results are summarized in Table 1. These data show that when NI 672 was introduced into the cell with fusion factor, no significant rise in the rate of successful infection over the background of “leaky” virus present in all NI 672 stocks was observed. For this experiment, it must be noted that C/A phenotype chick embryo cells were employed and that these cells are
A REPLICATION TABLE
DEFECTIVE
1
MUTANT
OF RSV
53
almost totally resistant to subgroup A viruses including ts 672. This was done to assure that the penetration observed after fusion factor treatment was independent of viral envelope-host receptor interaction. In this way, it was possible to exclude the background of “leaky” virus that is always present at titers of lo3 to 10’ FFU/ml in NI 672 preparations. Controls are shown in which the infections were performed with C/BDE phenotype cells which are fully susceptible to ts 672 and to the “leaky” virus present in NI 672. This control in fact demonstrates the importance of reducing the background through the use of resistant cells.
pears unlikely to demonstrate the particular lesion as a striking change in the rate of migration of a particular protein. The probability is much greater, however, that since a particular defective protein fails to function in its designated role in conferring infectivity, that it will also fail to associate correctly with other components at the time of virus maturation. If such were to be the case, analysis of the proteins of NI 672 compared to ts 672 might be expected to reveal quantitative differences in the amount of a particular protein incorporated into virus. It might be deficient, in excess, or totally absent. For this experiment, “C- and 3H-labeled amino acid mixtures of identical composition and relative activities were used so that a strict comparison of coelectrophoresed viral proteins would be possible. Figure 1 shows the result of the coelectrophoresis which failed to show any difference between NI 672 and ts 672. Similar coelectrophoresis between wild type PR-A grown at 41 C and NI 672 also indicated no aberration in the components of NI 672 (data not shown). Likewise, use of [“Cl- and [3H]glucosamine as precursors for labeling viral glycoproteins failed to indicate a structural defect in NI 672 (data not shown). Although there is always a low level of infectious virus in NI 672 preparations resulting from “leaky” function, this virus represents a fraction of less than 0.1% of the total present, and would not be expected to obscure real structural differences between NI 672 and ts 672. Therefore, while one cannot rule out the possibility of a subtle change in the structure of a protein which destroys its biological function without impairing its assembly into the viral structure, it seems likely that the structural proteins of NI 672 do not differ from those of ts 672.
The Structural
The RNA of NI 672
VIRAL PENETRATION MEDIATED BY SENDAI VIRUS FUSION FACTOR Virus
Cell phenotype
Observed titers (FFU/mlln Sendai treatment
Control
BH RSV( - I
C/BDE CIA
1 x loa 2 x lo3
