of an Infectious Form of Rous Sarcoma Virus*

Proceedings of the National Academy of Sciences Vol. 66, No. 2, pp. 314-321, June 1970 A Cell-Associated Factor Essential for Formation of an Infectious Form of Rous Sarcoma Virus* H. Hanafusa, T. Miyamoto, and T. Hanafusa THE PUBLIC HEALTH RESEARCH INSTITUTE OF THE CITY OF NEW YORK, INC. Communicated by George K. Hirst, March 9, 1970 Abstract. Formation of a specific type of infectious Rous sarcoma virus called RSV(O) has been found to occur only in a certain type of chick embryo cell in the absence of avian leukosis virus. Although these chick cells lack any complete form of avian leukovirus, they appear to carry a genetic factor essential for formation of infectious RSV(O). A factor-deficient cell can be converted to a producer of infectious RSV(O) by infection with Rous sarcoma virus or avian leukosis virus which has been grown in factor-containing cells. Evidence is presented to show that the factor determines both the antigenic and host range specificity of RSV(O). Cells infected and transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV)t produce RSV(O) in the absence of avian leukosis virus (ALV).1-5 The infectiousness of RSV(O) for certain avian host cells is conditioned by two factors: the genetic character of the virus and the type of host cells in which the RSV(O) was produced.5' 6 Thus, C/O-type chick embryo cells transformed by A-type Rous sarcoma virus release infectious RSVf3(0), whereas those transformed by the a-type variant produce noninfectious particles known as RSVa(O).5 However, even the RSVf3 cannot produce infectious Rous sarcoma virus when it infects C/O'-type cells and all transformants produce another noninfectious form called RSVf'(O).6 Unlike RSVa(O), RSVf'(O) differs from infectious RSVfl(O) only in its phenotype. Both RSVO(O) and RSV#'(O) can transform even naturally resistant chick cells with the aid of ultraviolet-irradiated Sendai virus, and the resulting transformed cells, if they are C/O cell type, produce RSVfl(O) regardless of which form of the RSV(O) was used to induce the transformation.6 Except for RSV3 (0), all avian leukoviruses7 (leukosis-sarcoma viruses) tested grow equally well in both C/O and C/O'-type cells. Thus, some mechanism specifically affecting the properties of RSV(O) must operate in these chick cells. Experiments described in this paper attempt to elucidate this mechanism. Evidence is presented for the presence in normal C/O cells of a genetic factor that shares some of the characteristics of avian leukoviruses. This factor is responsible for the formation of the infectious form of RSV(O) and can be transferred from C/O to C/O'-type cells by being carried by either the Rolls sarcoma or avian leukosis virus. Materials and Methods. Cell culture and viruses: The media and basic techniques for tissue culture of chick or quail embryo cells have been (lescribed previously.5 6, 8 Fertile chicken eggs were supplied by SPAFAS, Inc., Connecticut, and 314

VOL. 66, 1970 MICROBIOLOGY: HANAFUSA ET AL. 315) quail eggs were obtained from our own breeders. The Bryan high-titer strain of Rous sarcoma virus used in this study was the (3-type. Both RSV,3(O) and RSVB(ALV) were prepared from (3-type transformed cells.5'6 Their titers were assayed on quail and C/O chick cells, respectively, in the presence of DEAE-dextran,5 and expressed as focus-forming units for these cells. The procedure for infection of chick cells by RSV(3(0) with ultraviolet-inactivated Sendai virus is essentially the same as described before;6 cells were exposed to Rous sarcoma virus with ultraviolet-sendai virus (5000 hemagglutinating units) and DEAE-dextran (50,.g/ml), and seeded into plates after successive incubation at 00 and 370C. Definition and nomenclature of chick embryo cells: All chick embryo cells used were C/0 type according to the classification based on their genetic susceptibility to avian leukoviruses.9 These chick embryos were further differentiated by their susceptibility to RSV(3(0) and by their capacity to produce RSVJ#(0) after transformation by low doses of RSV(3(ALV) or RSV#(0). The characteristics of these subtypes are summarized in Table 1. For convenience, each subtype transformed by Rous sarcoma virus will be called r-(3-, r-(3'-, s-(3-, or s-(3'-transformed cells. TABLE 1. Characteristics of chick cells. Cells Form of RSV(0) Subtype of Susceptibility to transformed produced by the embryo RSV,6(0) RSV#(ALV) with RSV,6t transformed cells clof r-c/0 r* S r-fl- RSVfl(0)J s-c/o s* s s-ki- RSV,6(0) C/o' r-c/o' r s r-(3'- RSVfl'(0)J ls-c/o' s s s-,('- RSVI3'(0) * r: resistant, s: susceptible. 106 FFU of RSV#(0) form 105 to 106 foci on the susceptible cultures and no foci on the resistant ones. t These transformed cells are induced by low doses of RSVjB(ALV) or RSV,6(0) and, therefore, free from ALV. r-o'- and s-fl'-transformed cells produce no detectable amount of RSVB(O) as long as they are kept with normal r-c/o' or s-c/o' cells.6' "' I RSVjI(0) is infectious for quail and susceptible chick cells, and RSVjB'(0) is not infectious for any cell thus far tested. Antiserum for RSV(3(O) and virus neutralization: Four-week-old chickens were inoculated in both wing webs with 3 X 105 FFU of RSV(3(0). Tumors appeared in a majority of these chickens within 4 weeks after infection." Two out of 30 such chickens lroduced a high titer of antibody for RSV(3(0).10 For neutralization, 0.9 ml of virus was incubated with 0.1 ml of undiluted antiserum at 370C for 30 min. The anti-rsv(3(0) serum reduced the titer of RSV(3(0) from 105 to less than 102 FFU, but did not neutralize RSV(RAV-1), RSV(RAV-2), or RSV(RAV-50).1" The antiserum for RAV-2 was prepared by a method previously reported,'2 and completely neutralized 9 X 106 FFU of RSV(RAV-2), but had no effect on RSV(3(0). Results. Reproduction of RSVB(O) in s-c/o' cells: Of over 200 individual C/O-type chick embryos studied, 68% have been identified as r-c/o, 24% as r-c/o', 7% as s-c/o, and 1% as s-c/o'-type. Two embryos were recently characterized as s-c/o' type, and soon it became apparent that the reproduction of RSV(3(O) in this type of cells has an unusual characteristic. As s-c/o' cells, when transformed by less than 50 FFU of either RSV(ALV) or RSV,3(O), all cells derived from these embryos produced RSV(3'(0) (Table 2). Like r-o'- transformed cells,6 such transformants produced high titers of RSV(ALV) when superinfected with RAV-1 or RAV-2, and the progeny RSV#'(0) had the potential of producing foci on r-c/o cultures by infection together with ultraviolet-setidai virus. However, when the same s-c/o' cultures were infected with 106 FFU of RSVI3(O), they produced a high titer of RSV,3(O) (Table 2). This apparent

316 MICROBIOLOGY: HANAFUSA ET AL. PROC. N. A. S. TABLE 2. Efect of input virus concentration on RSV,3(O) production in s-c/o' cells.* Titer of Stock Chick input virus No. of No. of foci no. of embryo (FFU per foci producing Days Titer of RSVjB(O) no. culture) isolated RSVB(0) harvested RSVJI(O) M254 804 s-c/o' 35 20 0...... 0090 840 s-c/o' 40 19 0...... 0022 840 s-c/o' 50 13 0 M254 804 s-c/o' 3.3 X 106...... 11 1.0 X 105 M243 804 s-c/o' 9.8 X 106...... 13 7.9 X 106 B-81 840 s-c/o' 9.8 X 106...... 7 3.0 X 105 M254 825 r-c/o't 28 19 0...... M254-2t 825 r-c/o' 44 22 0...... * 1.2 X 106 chick embryo cells indicated were infected with either high or low doses of various preparations of RSVB(0). Foci were isolated from the cultures infected with low doses of virus, and production of RSV#(0) from each line containing more than 105 transformed cells was examined. When superinfected with RAV-1, all lines produced RSV(RAV-1) at an average titer of 4.4 X 106 FFU/ml. Cultures infected with high doses of RSV#(0) were subcultured once and the titer of RSVj9(0) in the culture fluid was determined at the day indicated. t M254-2 is the stock obtained from s-c/o' cells transformed with a high dose of M254. $ Infection of r-c/o' cells with RSVj3(0) was made in the presence of ultraviolet-inactivated Sendai virus. discrepancy between cultures infected with low and high doses of RSVf3(0) was reproducibly demonstrated with cells derived from the two embryos of this type and with three preparations of RSV,3(0) obtained from different r-f3-transformed cells. One explanation for this phenomenon may be the presence in every RSV,3(0) stock of a small amount of a mutant which is capable of growing in C/O' cells. Such a mutant virus might have been demonstrated only by infection with a large dose of RSVi(0). To test this possibility, the progeny RSVf3(0) recovered from s-c/o' cells transformed by 3 X 105 FFU of RSV,3(0) was inoculated into r-c/o' cells with ultraviolet Sendai virus. Twenty-two single foci were isolated from the infected cultures and production of RSV,3(0) from each line of transformed cells was examined. As a control, 19 foci were isolated from the same r-c/o' cultures infected with the parental RSVi3(0) in the same manner. None of the transformed cells formed by either parental or progeny RSVl3(0) produced RSVI3(0), indicating that selection of such a "mutant" virus had not occurred (Table 2). In order to obtain more quantitative information about the effect of virus dilution on production of infectious RSVf3(0) in s-c/o' cells, the s-c/o' cells were infected with serial dilutions of RSVj3(0) and the yield of RSV,3(0) from the cultures at various times after infection was determined. The results of this experiment (Fig. 1) confirmed the previous one; infectious RSV,3(0) was synthesized only in cultures infected with low dilutions of RSV43(0), and in those infected by less than 102 FFU of RSV,3(0) no RSV,3(0) was produced despite the complete transformation of the cultures after several transfers. It can also be seen that the time required before detectable RSV#(0) appeared in the cultures was highly dependent on the input virus concentration and, even with the highest multiplicity used, the production of RSV,8(0) in s-c/o' was much slower than that in s-c/o cells. Although we have failed to maintain quail transformed cells derived from single foci, the general characteristics of RSV,3(0) production in quail cells were very similar to that in s-c/o' cells: RSV,3(0) production occurred in quail trans-

VOL. 66, 1970 MICROBIOLOGY: HANAFUSA ET AL. 317 FIG. 1.-Production of infectious virus from s-c/0' cultures infected with varied 7 doses of RSV#(0). 1.2 X 106 S-C/0' cells were infected with 0.5 ml of serial ten-fold -106) dilutions of 1 X 105 FFU/ml of IRSV#(0), 5 (0, undiluted; A, 10-'; O], 10-2; V, 10-3), 1 (>5X105) and the infected cultures were transferred 6 every 4 days. The titer of RSV#3(0) in each >C 1 ) culture fluid was assayed on quail cells. As a i, x66xi05 (>06) control, a s-c/0 cell culture was infected with / undiluted RSVO(0) (@). The figure in the ' parentheses indicates the approximate num- (>1Q6) ber of transformed cells in each culture. (5X1044600) (3X05) (>106)(s106 T Points with arrows mean that infectious virus 4 8 2 16 2 24 was undetectable. 4 8 12 6204 DAYS AFTER INFECTION formed-cell cultures only when they had been infected with high doses of RSVI3- (0) Ṫhese results suggest that the stocks of RSV,8(O) consist of at least two different virus particles, and one of them cannot reproduce RSVf3(O) by itself in C/O' cells. This heterogeneity of virus may originate in C/G-type cells, since the stock viruses were obtained from these cells. Although other possibilities are conceivable, the above facts are consistent with the idea that an unidentified cellular or viral factor, which is present only in C/O cells and essential for formation of RSVI#(O) is transmitted from C/O to s-c/g' cells by being carried by a small fraction of RSV,8(0), resulting in formation of RSV,3(O) in the recipient cells. The results obtained in Figure 1 indicate that after transmission to s-c/g' cells, such a putative factor must multiply at least in the presence of Rous sarcoma virus genomes, at a rate probably faster than that of cell division. There is no evidence for the presence in C/O cells of avian leukosis virus of known type detectable either by labeling with a radioactive precursor of RNA, or by ability to induce formation of RSVf3(O) in s-3'-transformed cells which have been made by infection with low doses of RSV,3(O). However, most, if not all, tissues and culture cells of 47 C/O embryos thus far tested contained groupspecific antigen common to avian luekoviruses, and neither 15 C/O' nor 8 quail embryos had this antigen.'3 It is conceivable that the synthesis of groupspecific antigen, which is an internal component of virus, is another expression of the putative factor in C/O cells. Avian leukosis virus grown in C/O and C/O' cells: The following studies were performed to find out whether or not the factor can also be transferred from C/O to C/O' cells by other avian leukoviruses. Serial dilutions of RAV-2 were inoculated into both r-c/g and r-c/g' cells. Stocks of RAV-2 were obtained from the terminal dilutions of infected cultures after two transfers and designated as RAV-2 C/O and RAV-2- C/O', respectively. The two preparations of RAV-2 were then added to duplicate cultures of r-f3'-transformed cells, and the titer of Rous sarcoma virus in these cultures was assayed on both r-c/g and quail cells. In a separate experiment using antibodies to Rous sarcoma virus we had confirmed that, contrary to the case with r-c/g cells, quail cells are susceptible to RSVj(O) but completely resistant to RSV(RAV-2). Thus, one can determine

318 MICROBIOLOGY: HANAFUSA ET AL. Puoc. N. A. S. FIG. 2.-Productioii of i- SVO(O) fromr- #'-transformed 5 f I cells following infection with RAV-2. Two identical cultures of r-#'-transformed cells were 0> linfected with 107 infectious units a: 3 1 of either RAV-2.C/0' (A) or l RAV-2 0C/0 (B). The focusforming titers on r-c/0 cells are 0 24 48 0 24 48 HOURS AFTER INFECTION WITH RAV-2 for RSV(RAV-2) (A) and those on quail cells for RSV#(O) (0). the titer of these two kinds of Rous sarcoma virus independently on r-c/0 and quail cells. As shown in Figure 2, infection of r-f3'-transformed cells by RAV- 2. C/O' produced only RSV(RAV-2), whereas infection by RAV-2 0C/O resulted in formation of both RSV(RAV-2) and RSV#3(O). The results were confirmed with a separately prepared set of RAV-2 C/O, RAV-2 C/O', and five other lines of r-,3'-transformed cells. When RAV-2 C/0 and RAV-2 C/O' were passaged at terminal dilutions in the reverse type cells, i.e., r-c/o' and r-c/0 cells, the ability to produce RSVfl(O) from r-f3'-transformed cells was found only with RAV-2 passaged in r-c/o cells in the second passage. These experiments clearly demonstrate the presence of a factor in C/O cells which can be picked up and transmitted to C/O' cells by RAV-2. The results also indicate that the factorcarrying RAV-2 is a minor fraction in RAV-2 0C/O preparation. Formation of RSVJ3(O) from r-g'-transformed cells was also induced by RAV-2 0C/O purified through sucrose gradient centrifugation, indicating that the factor is transferred in a form of physically intact virus particles. Since the r-,3'-transformed cells are resistant to RSVf3(O), the above results show that the factor-containing particles demonstrated in these cells have RAV-2-specific envelope determinant. Role of a factor in C/O cells: The foregoing studies did not determine whether or not the factor is responsible for the synthesis of some structural component of RSVf3(O). The factor itself or its products could, for example, counteract a substance present in C/O' cells which represses the formation of infectious RSV,3(O). The synthesis of group-specific-antigen in C/O cells could be a direct cause of RSVI3(O) production, but the group-specific antigen itself was proved not to be deficient in RSVf3'(O) as well as in C/O' cells infected with RSV,8'(0).13 We have studied the envelope antigen of RSV,3(O) and RSVJ3'(O), and found that RSVj3'(O) lacks the capacity to absorb antibody against RSV,3(O).10 Although this does not exclude possible cross immunity between the two particles, it shows that the structure to their surface antigens is not identical. If the factor is directly involved in the synthesis of the surface of RSVYB(O), then avian leukosis virus grown in C/O cells may contain particles with the same coat antigen as RSVf3(O). RAV-2 -C/0 and RAV-2 0C/O' were treated with anti- RAV-2 alone or both anti-rav-2 and anti-rsv,3(o). The treated and untreated viruses were inoculated into r-c/o and s-c/0 cells after serial dilutions. The

VOL. 66, 1970 MICROBIOLOGY: HANAFUSA ET AL. 319 formation of RAV-2 in these cultures was examined both by interference with RSV(RAV-2) infection and by the ability of culture fluid to synthesize RSV- (RAV-2) in r-8--transformed cells. The results are summarized in Table 3. TABLE 3. Demtionstration of RSV#(O)-plheotypic characteristics in R. V-2.* Highest Dilution Which Produced RAV-2 Following Infection on Virus Treated with r-c/o s-c/o - RAV-2 C/O Untreated 10-6 10-6 RAV-2 * C/O Anti-RAV-2 Undetectablet 10-1 RAV-2. C/O Anti-RAV-2+anti-RISV,8(0) Undetectable Undetectable RAV-2 C/O' Untreated 10-6 10-6 RAV-2 C/0)' Anti-RAV-2 Undetectable Undetectable * RAV-2 grown in either r-c/o or r-c/o' cells was mixed with anti-rav-2 or anti-rav-2 plus anti-rsv#(o). Serial ten-fold dilutions of the treated and untreated viruses were made and 0.1 ml of each dilution was inoculated into both r-c/o and s-c/o cells. The cultures were transferred twice and the presence of RAV-2 in each culture was detected by two methods described in the text. Both methods gave identical results. t The cultures infected with 0.1 ml of undiluted samples gave negative results for RAV-2. The complete neutralization of RAV-2. C/O' by anti-rav-2 was demonstrated by failure in RAV-2 production by the treated virus in both r-c/0 and s-c/o cells. The RAV-2. C/O treated with anti-rav-2 also completely lost its infectivity for r-c/o cells, but still retained infectivity for s-c/o cells, as shown by RAV-2 production in the latter cells. This residual infectivity for s-c/0 cells was, however, neutralized by anti-rsvb(o) serum. Therefore, unlike RAV- 2. C/0', which consists of a homogeneous population of virus with RAV-2 antigenicity, RAV-2 *C/O also contains RAV-2 particles with the envelope specific for RSVB(O) in characteristics of both host range and surface antigen. From these results we concluded that this factor in C/O cells is responsible for formation of an envelope component of RSVB (0) which probably is essential for the infectiousness of RSVB(O). Discussion. Although RSV(O) shares common physical and biological characteristics with Rous sarcoma virus produced in the presence of avian leukosis virus,i-5 it notably differs from other members of leukoviruses in its host range. The uniqueness of RSV(O) is further exemplified by its dependence on certain types of host cells for reproduction of infectious progeny.6 In explaining this host dependence of virus reproduction, we have suggested the mechanism of hostcontrolled modification in certain chick cells.6 The fact that the factor transmitted to C/G' cells by RSV and ALV enable RSV to mature into infectious virus in the recipient cells rules out host controlled modification as the mechanism for formation of RSV,8(0). The factor has some similarity to ALV as a helper for RSV,14 since it appears to determine both the antigenicity and host range specificity of RSVJ3(O). However, the factor does not appear to exist in the cells as a complete form of ALV, at least before infection with avian leukovirus. We propose to call this factor "chick cell-associated helper factor (chf or f)," and define it as a genetic entity which assists formation of one infectious form of Rous sarcoma virus.'5 In view of the presence in C/O cells of the group-specific antigen which may well be coded for by the chf, the chf does not seem to be in a repressed state as the "oncogene" proposed by Huebner and Todaro.'6 In

320 MICROBIOLOGY: HANAFUSA ET AL. PROC. N. A. S. attempts to gain further evidence, studies are also being made to find out whether or not the surface antigen for RSVfl(O) is present in the C/O cells. While the finding of the chf provides an explanation for the formation of RSV,3(O) in C/O cells, it still does not explain the unusual host range of this virus. For this reason, the mechanisms for formation of infectious Rous sarcoma virus by the chf and by avian leukosis virus may not be entirely the same. The presence of the group-specific antigen in some apparently normal chick embryos has been described.'7 18 A recent report by Weiss'9 that the formation of infectious RSV# (0) occurs only in the cells derived from a chicken line possessing the group-specific antigen is consistent with our findings.'3 From genetic studies with chicken strains with or without the group specific-antigen, Payne and Chubb 18 demonstrated that the formation of this antigen is most likely a characteristic of cells which may be inherited by the descendants through a single dominant autosomal gene in a simple Mendelian manner. If this is also the case in the chick cells used in this study, and if the group-specific antigen is another product of the chf, then one must assume that the genetic information of the chf is maintained in the cells in a form associated with the host cell chromosomes. However, if we define the chf as an entity transmissible by avian leukoviruses from one cell to another, it is probably in a form of RNA rather than DNA. Perhaps, even if its integrated form is associated with the host DNA, the chf could be present in the cells as RNA. Studies on the chemical nature and intracellular state of the chf are needed to provide information on these problems. Quantitative aspects of the interaction of the chf with Rous sarcoma virus and avian leukosis virus will be given elsewhere. The results described in this paper show that less than 10-3 of either RSV or ALV particles carry the chf. It remains to be determined whether the chf is packed into these particles independently or as part of the RSV or ALV genomes. The presence of factor has been demonstrated only in the C/O-type chick cells. It is conceivable that a similar factor is present in other genetic types of chick cells or even in other species of animals. The modulation of murine and avian sarcoma virus reported by Klement et al.20 and Altaner and Temin" might be related to the presence of such a factor in some mammalian host cells. The authors wish to thank Susan Zanger, Lucy DiCicco, and Judith Leichtberg for their excellent technical assistance. Requests for reprints should be addressed to Dr. H. Hanafusa, The Public Health Research Institute of the City of New York, Inc., 455 First Avenue, New York, New York 10016. * This investigation was supported in part by the U.S. Public Health Service research grant CA 08747 from the National Cancer Institute. t Abbreviations used: RSV, Rous sarcoma virus; ALV, avian leukosis virus; RAV, Rousassociated virus; FFU, focus-forming unit; chf, chick cell-associated helper factor; D)EAE-, diethyl-aminoethyl-. ' Dougherty, R. M., and H. S. Di Stefano, Virology, 27, 351 (1965). 2 Robinson, H. L., these PROCEEDINGS, 57, 1655 (1967). 3Weiss, R. S., Virology, 32, 719 (1967). 4 Vogt, P. K., these PROCEEDINGS, 58, 801 (1967). 5 Hanafusa, H., and T. Hanafusa, Virology, 34, 630 (1968). 6 Hanafusa, T., T. Miyamoto, and H. Hanafusa, Virology, 40, 55 (1970). 7Fenner, F., in The Biology of Animal Viruses (New York: Academic Press, 1968), p. 26. 8 Hanafusa, H., these PROCEEDINGS, 63, 318 (1969).

VOL. 66, 1970 MICROBIOLOGY: HANAFUSA ET AL. 321 9 Vogt, P. K., and R. Ishizaki, Virology, 26, 664 (1965). 10 Miyamoto, T., T. Hanafusa, and H. Hanafusa, manuscript in preparation. 11 The chickens used were raised from fertile eggs derived from the same flock as those used for cell cultures, the majority of which are not susceptible to RSV#(0). Therefore, the high incidence (93%) of tumor induction by RSVI3(0) in chickens was an unexpected finding.'0 12 Hanafusa, H., T. Hanafusa, and H. Rubin, these PROCEEDINGS, 51, 41 (1964). 13 Miyamoto, T., E. Fleissner, and H. Hanafusa, manuscript in preparation. 14 Hanafusa, H., T. Hanafusa, and H. Rubin, these PROCEEDINGS, 49, 572 (1963); Hanafusa, H., Virology, 25, 248 (1965). "I The term RSV(0) may not be appropriate in view of the finding of the chf. RSV,8(0) may be called RSVfI (f) according to the nomenclature customarily adopted for RSV(ALV) produced in the presence of ALV.12, 14 The chf-carrying RSV particles may be indicated by RSV#- f(f). Likewise, the present results show that RAV-2 grown in C/O cells contains at least three kinds of particles, namely RAV-2(RAV-2), RAV-2(f), and RAV-2.f(RAV-2). Further studies on these particles will prove the adequacy of these terms. ' Huebner, R. J., and G. J. Todaro, these PROCEEDINGS, 64, 1087 (1969). 17 Dougherty, R. M., and H. S. Di Stefano, Virology, 29, 586 (1966). 18 Payne, L. N., and R. C. Chubb, J. Gen. Virol., 3, 379 (1968). 19 Weiss, R. A., J. Gen. Virol., 5, 511 (1969). In this paper, the author used the term RSVa- (0), which was proposed by us for a genetic variant of RSV#(0), for noninfectious particles which are similar in every respect to RSVjB'(0). Thus, "RSVa(O)" and "L-Ra" as used by this author should be equivalent to RSVg3'(0) and B'-type transformed cells by our definition.". 6 20 Klement, V., J. W. Hartley, W. Rowe, and R. J. Huebner, J. Natl. Cancer Inst., 43, 925 (1969). 21 Altaner, C., and H. M. Temin, Virology, 40, 118 (1970).