Proceedings of the National Academy of SciencesVol. 66, No. 2, pp. 314-321, June 1970

A Cell-Associated Factor Essential for Formationof an Infectious Form of Rous Sarcoma Virus*

H. Hanafusa, T. Miyamoto, and T. HanafusaTHE 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 calledRSV(O) has been found to occur only in a certain type of chick embryo cell inthe absence of avian leukosis virus. Although these chick cells lack any com-plete form of avian leukovirus, they appear to carry a genetic factor essentialfor formation of infectious RSV(O). A factor-deficient cell can be convertedto a producer of infectious RSV(O) by infection with Rous sarcoma virus oravian leukosis virus which has been grown in factor-containing cells. Evidenceis presented to show that the factor determines both the antigenic and hostrange specificity of RSV(O).

Cells infected and transformed by the Bryan high-titer strain of Rous sarcomavirus (RSV)t produce RSV(O) in the absence of avian leukosis virus (ALV).1-5The infectiousness of RSV(O) for certain avian host cells is conditioned by twofactors: the genetic character of the virus and the type of host cells in which theRSV(O) was produced.5' 6 Thus, C/O-type chick embryo cells transformed byA-type Rous sarcoma virus release infectious RSVf3(0), whereas those transformedby the a-type variant produce noninfectious particles known as RSVa(O).5However, even the RSVf3 cannot produce infectious Rous sarcoma virus when itinfects C/O'-type cells and all transformants produce another noninfectious formcalled 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 nat-urally resistant chick cells with the aid of ultraviolet-irradiated Sendai virus, andthe resulting transformed cells, if they are C/O cell type, produce RSVfl(O) re-gardless of which form of the RSV(O) was used to induce the transformation.6Except for RSV3 (0), all avian leukoviruses7 (leukosis-sarcoma viruses) tested growequally well in both C/O and C/O'-type cells. Thus, some mechanism specifi-cally 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 thatshares some of the characteristics of avian leukoviruses. This factor is responsi-ble for the formation of the infectious form of RSV(O) and can be transferred fromC/O to C/O'-type cells by being carried by either the Rolls sarcoma or avianleukosis virus.

Materials and Methods. Cell culture and viruses: The media and basic teCh-niques for tissue culture of chick or quail embryo cells have been (lescribed pre-viously.5 6, 8 Fertile chicken eggs were supplied by SPAFAS, Inc., Connecticut, and

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quail eggs were obtained from our own breeders. The Bryan high-titer strain of Roussarcoma virus used in this study was the (3-type. Both RSV,3(O) and RSVB(ALV) wereprepared from (3-type transformed cells.5'6 Their titers were assayed on quail and C/Ochick cells, respectively, in the presence of DEAE-dextran,5 and expressed as focus-form-ing units for these cells. The procedure for infection of chick cells by RSV(3(0) withultraviolet-inactivated Sendai virus is essentially the same as described before;6 cells wereexposed to Rous sarcoma virus with ultraviolet-Sendai virus (5000 hemagglutinatingunits) and DEAE-dextran (50 ,.g/ml), and seeded into plates after successive incuba-tion at 00 and 370C.

Definition and nomenclature of chick embryo cells: All chick embryo cells usedwere C/0 type according to the classification based on their genetic susceptibility toavian leukoviruses.9 These chick embryos were further differentiated by their suscepti-bility to RSV(3(0) and by their capacity to produce RSVJ#(0) after transformation by lowdoses of RSV(3(ALV) or RSV#(0). The characteristics of these subtypes are summarizedin Table 1. For convenience, each subtype transformed by Rous sarcoma virus will becalled 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 theembryo RSV,6(0) RSV#(ALV) with RSV,6t transformed cells

clofr-C/0 r* S r-fl- RSVfl(0)Js-C/O s* s s-ki- RSV,6(0)

C/o' r-C/O' r s r-(3'- RSVfl'(0)Jls-C/O' s s s-,('- RSVI3'(0)

* r: resistant, s: susceptible. 106 FFU of RSV#(0) form 105 to 106 foci on the susceptible culturesand 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 longas 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 forany cell thus far tested.

Antiserum for RSV(3(O) and virus neutralization: Four-week-old chickens wereinoculated in both wing webs with 3 X 105 FFU of RSV(3(0). Tumors appeared in amajority of these chickens within 4 weeks after infection." Two out of 30 such chickenslroduced a high titer of antibody for RSV(3(0).10 For neutralization, 0.9 ml of virus wasincubated 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 neutralizeRSV(RAV-1), RSV(RAV-2), or RSV(RAV-50).1" The antiserum for RAV-2 was pre-pared by a method previously reported,'2 and completely neutralized 9 X 106 FFU ofRSV(RAV-2), but had no effect on RSV(3(0).

Results. Reproduction of RSVB(O) in s-C/O' cells: Of over 200 individualC/O-type chick embryos studied, 68% have been identified as r-C/O, 24% asr-C/O', 7% as s-C/O, and 1% as s-C/O'-type. Two embryos were recentlycharacterized as s-C/O' type, and soon it became apparent that the reproduc-tion 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) whensuperinfected with RAV-1 or RAV-2, and the progeny RSV#'(0) had the potentialof producing foci on r-C/O cultures by infection together with ultraviolet-Seti-dai virus. However, when the same s-C/O' cultures were infected with 106 FFUof RSVI3(O), they produced a high titer of RSV,3(O) (Table 2). This apparent

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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 focino. 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 0M254 804 s-C/O' 3.3 X 106 ... ... 11 1.0 X 105M243 804 s-C/O' 9.8 X 106 ... ... 13 7.9 X 106B-81 840 s-C/O' 9.8 X 106 ... ... 7 3.0 X 105M254 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 variouspreparations 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 106FFU/ml. Cultures infected with high doses of RSV#(0) were subcultured once and the titer ofRSVj9(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) wasreproducibly demonstrated with cells derived from the two embryos of thistype and with three preparations of RSV,3(0) obtained from different r-f3-trans-formed cells. One explanation for this phenomenon may be the presence in everyRSV,3(0) stock of a small amount of a mutant which is capable of growing inC/O' cells. Such a mutant virus might have been demonstrated only by infec-tion with a large dose of RSVi(0). To test this possibility, the progenyRSVf3(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 singlefoci were isolated from the infected cultures and production of RSV,3(0) from eachline of transformed cells was examined. As a control, 19 foci were isolated fromthe 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 notoccurred (Table 2).

In order to obtain more quantitative information about the effect of virus dilu-tion on production of infectious RSVf3(0) in s-C/O' cells, the s-C/O' cells wereinfected with serial dilutions of RSVj3(0) and the yield of RSV,3(0) from thecultures at various times after infection was determined. The results of thisexperiment (Fig. 1) confirmed the previous one; infectious RSV,3(0) was synthe-sized only in cultures infected with low dilutions of RSV43(0), and in those infectedby less than 102 FFU of RSV,3(0) no RSV,3(0) was produced despite the completetransformation of the cultures after several transfers. It can also be seen that thetime required before detectable RSV#(0) appeared in the cultures was highlydependent on the input virus concentration and, even with the highest multiplicityused, the production of RSV,8(0) in s-C/O' was much slower than that in s-C/Ocells. Although we have failed to maintain quail transformed cells derived fromsingle foci, the general characteristics of RSV,3(0) production in quail cells werevery similar to that in s-C/O' cells: RSV,3(0) production occurred in quail trans-

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FIG. 1.-Production of infectious virusfrom s-C/0' cultures infected with varied 7doses of RSV#(0). 1.2 X 106 S-C/0' cellswere 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 6every 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)(s106Points with arrows mean that infectious virus T4 8 2 16 2 24was undetectable.

4 8 12 6204

DAYS AFTER INFECTION

formed-cell cultures only when they had been infected with high doses of RSVI3-(0).These 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 stockviruses were obtained from these cells. Although other possibilities are con-ceivable, the above facts are consistent with the idea that an unidentified cellularor viral factor, which is present only in C/O cells and essential for formation ofRSVI#(O) is transmitted from C/O to s-C/G' cells by being carried by asmall fraction of RSV,8(0), resulting in formation of RSV,3(O) in the recipientcells. The results obtained in Figure 1 indicate that after transmission tos-C/G' cells, such a putative factor must multiply at least in the presence ofRous 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, orby ability to induce formation of RSVf3(O) in s-3'-transformed cells which havebeen 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 group-specific antigen common to avian luekoviruses, and neither 15 C/O' nor 8 quailembryos had this antigen.'3 It is conceivable that the synthesis of group-specific antigen, which is an internal component of virus, is another expression ofthe 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 fromC/O to C/O' cells by other avian leukoviruses. Serial dilutions of RAV-2 wereinoculated into both r-C/G and r-C/G' cells. Stocks of RAV-2 were obtainedfrom the terminal dilutions of infected cultures after two transfers and desig-nated as RAV-2 C/O and RAV-2-C/O', respectively. The two preparations ofRAV-2 were then added to duplicate cultures of r-f3'-transformed cells, and thetiter of Rous sarcoma virus in these cultures was assayed on both r-C/G and quailcells. In a separate experiment using antibodies to Rous sarcoma virus we hadconfirmed that, contrary to the case with r-C/G cells, quail cells are susceptible toRSVj(O) but completely resistant to RSV(RAV-2). Thus, one can determine

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FIG. 2.-Productioii ofi- SVO(O) fromr- #'-transformed

5 f I cells following infection withRAV-2. Two identical culturesof r-#'-transformed cells were

0> linfected with 107 infectious unitsa: 3 1 of either RAV-2.C/0' (A) or

l RAV-2 0C/0 (B). The focus-forming titers on r-C/0 cells arefor RSV(RAV-2) (A) and thoseon quail cells for RSV#(O) (0).

0 24 48 0 24 48HOURS AFTER INFECTION WITH RAV-2

the titer of these two kinds of Rous sarcoma virus independently on r-C/0 andquail 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 resultedin formation of both RSV(RAV-2) and RSV#3(O). The results were confirmedwith a separately prepared set of RAV-2 C/O, RAV-2 C/O', and five other linesof r-,3'-transformed cells. When RAV-2 C/0 and RAV-2 C/O' were passagedat terminal dilutions in the reverse type cells, i.e., r-C/O' and r-C/0 cells, theability to produce RSVfl(O) from r-f3'-transformed cells was found only withRAV-2 passaged in r-C/O cells in the second passage. These experiments clearlydemonstrate the presence of a factor in C/O cells which can be picked up andtransmitted to C/O' cells by RAV-2. The results also indicate that the factor-carrying RAV-2 is a minor fraction in RAV-2 0C/O preparation. Formation ofRSVJ3(O) from r-g'-transformed cells was also induced by RAV-2 0C/O purifiedthrough sucrose gradient centrifugation, indicating that the factor is transferredin a form of physically intact virus particles. Since the r-,3'-transformed cells areresistant to RSVf3(O), the above results show that the factor-containing particlesdemonstrated 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 ofRSVf3(O). The factor itself or its products could, for example, counteract a sub-stance 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 ofRSVI3(O) production, but the group-specific antigen itself was proved not to bedeficient in RSVf3'(O) as well as in C/O' cells infected with RSV,8'(0).13 We havestudied 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 notexclude possible cross immunity between the two particles, it shows that thestructure to their surface antigens is not identical.

If the factor is directly involved in the synthesis of the surface of RSVYB(O), thenavian leukosis virus grown in C/O cells may contain particles with the same coatantigen 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 untreatedviruses were inoculated into r-C/O and s-C/0 cells after serial dilutions. The

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formation of RAV-2 in these cultures was examined both by interference withRSV(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 ProducedRAV-2 Following Infection on

Virus Treated with r-C/O s-C/ORAV-2 - C/O Untreated 10-6 10-6RAV-2 * C/O Anti-RAV-2 Undetectablet 10-1RAV-2. C/O Anti-RAV-2+anti-RISV,8(0) Undetectable UndetectableRAV-2 C/O' Untreated 10-6 10-6RAV-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 plusanti-RSV#(O). Serial ten-fold dilutions of the treated and untreated viruses were made and 0.1 mlof each dilution was inoculated into both r-C/O and s-C/O cells. The cultures were transferredtwice 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 demonstratedby failure in RAV-2 production by the treated virus in both r-C/0 and s-C/Ocells. The RAV-2 .C/O treated with anti-RAV-2 also completely lost its infec-tivity for r-C/O cells, but still retained infectivity for s-C/O cells, as shownby RAV-2 production in the latter cells. This residual infectivity for s-C/0 cellswas, however, neutralized by anti-RSVB(O) serum. Therefore, unlike RAV-2. C/0', which consists of a homogeneous population of virus with RAV-2antigenicity, RAV-2 *C/O also contains RAV-2 particles with the envelope spe-cific for RSVB(O) in characteristics of both host range and surface antigen. Fromthese results we concluded that this factor in C/O cells is responsible for formationof an envelope component of RSVB(0) whichprobably is essential for the infectious-ness of RSVB(O).

Discussion. Although RSV(O) shares common physical and biological charac-teristics with Rous sarcoma virus produced in the presence of avian leukosisvirus,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 certaintypes of host cells for reproduction of infectious progeny.6 In explaining thishost dependence of virus reproduction, we have suggested the mechanism of host-controlled modification in certain chick cells.6 The fact that the factor trans-mitted to C/G' cells by RSV and ALV enable RSV to mature into infectious virusin the recipient cells rules out host controlled modification as the mechanism forformation of RSV,8(0). The factor has some similarity to ALV as a helper forRSV,14 since it appears to determine both the antigenicity and host rangespecificity of RSVJ3(O). However, the factor does not appear to exist in the cellsas a complete form of ALV, at least before infection with avian leukovirus. Wepropose to call this factor "chick cell-associated helper factor (chf or f)," anddefine it as a genetic entity which assists formation of one infectious form ofRous sarcoma virus.'5 In view of the presence in C/O cells of the group-specificantigen which may well be coded for by the chf, the chf does not seem to be in arepressed state as the "oncogene" proposed by Huebner and Todaro.'6 In

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attempts to gain further evidence, studies are also being made to find out whetheror not the surface antigen for RSVfl(O) is present in the C/O cells. While thefinding of the chf provides an explanation for the formation of RSV,3(O) in C/Ocells, 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 byavian 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 formationof infectiousRSV# (0) occurs only in the cells derived from a chicken line possessingthe group-specific antigen is consistent with our findings.'3 From genetic studieswith chicken strains with or without the group specific-antigen, Payne and Chubb 18demonstrated that the formation of this antigen is most likely a characteristic ofcells which may be inherited by the descendants through a single dominantautosomal gene in a simple Mendelian manner. If this is also the case in thechick cells used in this study, and if the group-specific antigen is another productof the chf, then one must assume that the genetic information of the chf is main-tained in the cells in a form associated with the host cell chromosomes. How-ever, if we define the chf as an entity transmissible by avian leukoviruses from onecell 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 bepresent in the cells as RNA. Studies on the chemical nature and intracellularstate of the chf are needed to provide information on these problems.

Quantitative aspects of the interaction of the chf with Rous sarcoma virus andavian leukosis virus will be given elsewhere. The results described in this papershow that less than 10-3 of either RSV or ALV particles carry the chf. It re-mains to be determined whether the chf is packed into these particles indepen-dently or as part of the RSV or ALV genomes. The presence of factor has beendemonstrated only in the C/O-type chick cells. It is conceivable that a similarfactor is present in other genetic types of chick cells or even in other species ofanimals. The modulation of murine and avian sarcoma virus reported byKlement et al.20 and Altaner and Temin" might be related to the presence of sucha factor in some mammalian host cells.

The authors wish to thank Susan Zanger, Lucy DiCicco, and Judith Leichtberg for theirexcellent technical assistance.

Requests for reprints should be addressed to Dr. H. Hanafusa, The Public Health ResearchInstitute 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 grantCA 08747 from the National Cancer Institute.

t Abbreviations used: RSV, Rous sarcoma virus; ALV, avian leukosis virus; RAV, Rous-associated 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).

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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 highincidence (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) producedin 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 kindsof particles, namely RAV-2(RAV-2), RAV-2(f), and RAV-2.f(RAV-2). Further studies onthese 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 particleswhich are similar in every respect to RSVjB'(0). Thus, "RSVa(O)" and "L-Ra" as used bythis author should be equivalent to RSVg3'(0) and B'-type transformed cells by our defini-tion.". 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).

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