Rev.Inst. Adolfo Lutz,49(2): 169-178, 1989.

EXPRESSION OF AVIAN LEUKOSIS VIRUS PROTEINS p27 AND p19 INUNINFECTED CHICKEN, DRAKE AND QUAIL CELLS

Júlia Maria Martins de SOUZA-FELIPPE*Tomoko HIGUCHI**

RIALA6/675

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avian leukosis virusproteins p27 and p19 in uninfected chicken, drake and quail cells. Rev. Inst,Adolfo Lutz, 49(2):169-178, 1989.

ABSTRACT: Normal, uninfected animal cells have an unique feature, the presencein their genome of genes (proviruses) identical or closely related to infectiousexogenous retrovirus. The level of expression of these genes varies from individual toindividual; from complete silence to the production of virus particles. Exogenous andendogenous virus proteins were purified from sonicated viruses passed through aSepharose 6B column satured with 6M guanidinium hydrochloride. The presence ofequivalents of antigens p27 and p19 was analysed by radioimmunoassay. Thepresence of endogenous virus components was investigated in CEP, heart, brain, tighmuscle and wing muscle of chicken, quail and drake. The function or significance ofthe proteins are not known. Immunological, hormonal and environmental factorsremain to be studied.

DESCRIPTORS: Retrovirus; Endogenous virus; virus endogenous expression; RIA.

INTRODUCTION

The retrovirus exhibits an unique feature, theircomplete genome or some of their genes mightbe present in uninfected, normal cells. This chro-mosome segment is named endogenous and it iswidely distributed in nature, having been charac-terized not only in birds but also in mammals.Its existence was first reported by DARLINGTON,194815 as provirus, a cellular genetic element ac-tivated under special circumstances. More carefullstudies conceming the presence of informationspecifying the genome of an RNA tumor virus innormal cells were carried out by HUEBNER &TODAR031. The virus specific DNA was proposedto be vertically transmitted and portions of lhegenome were proposed to be expressed duringcertain stages of the development. The authorsassumed that in normal adult cells the transcrip-tion of lhe genes would be repressed.

HANAFUSA et al.2° reported that fibroblastsfrom certain normal chicken embryos were ableto complement lhe defectiveness of the RSVBryan strain releasing infectious particles. Thefibroblasts contained one component, then calledchicken helper factor (chf), later on characterizedas an envelope glycoprotein encoded by endoge-nous viruses present in these cells. VOGT &FRISS59 found espontaneous release of RAV-O vi-ruses from certain chicken embryo cell lines.This observation led to the conclusion that theendogenous genome by itself is certainly defec-tive: these studies were corroborated by severalauthors 6.20.42.48.58.Later on, the presence of this en-velope glycoprotein was detected in lhe majorityof commercial flocks of chicken. Due to the ubi-quitous presence of the glycoprotein, it was sug-gested to be harrnless to the host organism andeven be advantageous in certain cases by protect-ing lhe host against exogenous infcctionw.

* Do Laboratório de Biologia Molecular, Virologia, Instituto Adolfo Lutz, São Paulo, SP.** Do Departamento de Bioquímica, Instituto de Química, USP, São Paulo, SP.

169

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avíanIeukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev, Inst. Adolfo Lutz, 49(2):169-178, 1989.

In regard to the presence of the helper factorand group specific antigen (gs), HANAFUSA etal,22 and ROBINSON47 identified three differentphenotypes in chicken embryos: gs+chf+ show-ing both properties; genetic experiments showedthe segregation of two single dominant alleles ;gs" - chi'" or h-e do not show detectable levelsof gs but high levels of helper acuvity:and gs - chf - which show either very low or unde-tectable level of both characters. Hybridizationexperiments conducted with specific probesshowed the absence in the genome of U3-regionrelated to exogenous virus in the chicken fibro-blasts analyscd=. Curiosly, the cells containingenv-3 do not express the structural viral cornpo-nent p15. So far, it has been recognized 16 env-endogenous species.

The distribution of the endogenous virus dif-fers tremendously in different species as well asin the same species. Moreover, the phenotypeexpression covers a wide range, going fromcomplete silence, expression of one componentuntil the production of a full particle. The expla-nation of this phenomenon is not known, al-though some hypothesis have been postulated,like the difference in the leveI of expressionmight be due to provirus mutation or chemicalmodification of the provirus genome. Methyla-tion has been shown to be an important modifi-cation12.23.33.S\the relevance of the pIOcess wasshown by using 5-azocitidine. JONES & TAY-LOR35; GROUDINE et al.19proposed the methyla-tion as a cell mechanism to repress the expres-sion of undesirable genes.

Hybridization experiments have shown thepresence of analogous retrovirus genes in germlines of primates. VARMUS & LEVINE (1983)56raised several questions about endogenousviruses. "Do they arise frorn normal cellulargenes? Are they derived in different speciesfrorn a primordial endogenous provirus in anancestral vertebrate? Do they find their wayinto germ lines by intermitent, independentinfectious? What are the benefits and dangers toa host organism that carries endogenousproviruses?"

In the present work, we have tried the purifi-cation of endogenous virus antigens and theircomparison with the pIOfiles obtained with ex-ogenous virus particles. The approach used tomeasure the expression of these protein was theradioimmunoassay. The expression of endoge-nous virus components mainly, p27 and p19,were studied in chicken embryo fibroblasts andextracts of chicken tissues and organs. Quail anddrake cellular extracts were also analyzed and theidentity of p27 and p19 in different systems wasinvestigated.

170

MATERIAL AND METHODS

Virus purification

Avian fibroblasts culture supernatants wereclarified by low speed centrifugation (3,500 xg/30 min./4°C); the supematants were submitted toultracentrifugation (100,000 xg/90min/4°C) toseparate virus particles. The pellet resuspendedin PBS (phosphate saline buffer = 120 mM sodi-um chloride; 18 mM disodium phosphate and 2.5mM kalium monobasic phosphate) was layeredonto a 20 to 60% sucrose-T'Rl S (10 mM pH 7.4)gradient. After centrifugation at 100,000 xg/10°C for 180 minutes, the fractions of the gradi-ent were collected and these fractions were recen-trifuged at 110,000 xg/90min/4°C. The com-bined pellets were resuspended in PBS anddialysed against buffer containing 40 mMTRIS.HC1 pH 7.4; 20 mM sodium acetate and1mMEDTA.

Isolation and purificaiion of structural proteins

Virus particles were disrupted in the presenceof TNE-TRITON X-100 0.2% by sonication andthe sample was suspended in buffer containing8M guanidine-hydrochlor ide: 1.5 M beta-mercaptoethanol and 5 mM EDTA pH 8.0. Thevirus extract was applied to a column of Sepha-rose 6B-guanidine hydrochloride and eluted withbuffer containing 50 mM sodium acetate pH 5.0;20 mM beta-mercaptoethanol and 6 M guanidinehydrochloride according to the procedure previ-ously described by GREEN & BOLOGNESP8.

Protein determination

The protein was determined either by LOWRY etaJ.38method or BRADFORD9 for samples contain-ing guanidine hydrochloride. Bovine serum albu-min was used as standard based on E~= 6.6.

Polyacrylamide sodium dodecyl sulphate gelelectrophoresis (PACE)

The gel electrophoresis system used were ei-ther at 12.5% polyacrylamide concentration or5-20% gradient according to the technique de-scribed by LAEMMLI36 and MAIZEL4o. High andlow molecular weight standards were used asmarkers.

Cell lines, tissues and extracts preparation

CEF-commercially available chicken embryofibroblasts culture; CEF-7.2-chicken embryo fib-roblasts line 7 subline 2; brain, heart, tigh andwing muscle from 5 months old chicken (Callusgallus domesticus), 30 days old quail (Nothura mac-ulosa) and 30 days old drake (Anas platyhynchos).

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avian leukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev, Inst. Adolfo Lutz, 49(2):169-178, 1989.

The tissues and organs were removed immedi-ately after bird killing: the fat was taken out andthe tis sues and organs werekept in an ice bath.The material was minced and homogenized in thepresence of TNE buffer containing 1mM PMSF(phenylmethylsulfonilfluoride) and TRITON X-100 0.2%. The excess of debris was discard andthe supernatant centrifuged at 1,500 xg/30min./4°C and then 10,000 xg/60min./4°C. To clean upthe extracts, the supernatants were centrifuged at80,000xg/60min/4°C and the final supernatantswere used as "cellular extracts" in the assays.

Radioimmunoassay

The viral and to procedures purified proteinswere labelled with 1251_Na according to HUN-TER34, according to technique described elsewhere27.28. These techniques allowed the time of reac-tion to be reduced to 30 seconds.

Immune serum titre - The titre was defined asthe serum dilution giving 50% precipitation oflabelled antigen under specified conditions. Con-stant amount of labelled antigen plus several di-lutions of immune serum plus normal rabbit ser-um were incubated at 37°C for 3 hours and thereaction completed at 4°C overnight, then theprecipitating sheep antibodies or goat anti-rabbit 19G were added. The reaction was allowedto precipitate at 4°C/overnight and subsequentlysubmitted to centrifugation at 3,500 xg!20min:the pellet was washed twice with cold TNE andthe radioactivity of the pellet measured in agamma counter.

Competition assay - The competition assay ofthe proteins of cellular extracts were performed ina system similar to the precipitation reaction,with the addition of cellular extracts. The amountof the equivalent of antigen present in the cellularextract was calculated by comparison with a stan-dard competition curve prepared by using knownamounts of unlabelled protein as competitor inthe above experimental conditions.

RESULTS

Purification of viruses particles

The virus particles separeted from culturefluids when submitted to sucrose gradient ultra-centrifugation showed different banding patternsdepending whether the virus, was exogenous orendogenous. The exogenous viruses formed onlyone layer around the sucrose concentration of35%, which is equivalent to a density of 1.16 -1.18g/mI7,8. The endogenous viruses presentedbesides the main layer at the concentration of35%, second band at around 20% and a third

layer (lower) between the sucrose concentrationof 50 and 60%. In addition, both endogenous vi-ruses 1515 and 7.2 presented similar protein pro-files in PAGE on a gel gradient 5 to 20% (figure1). Compared with the molecular weights mark-ers, 200 K = myosin; 150 K = immunoglobulin;90 K = phosphorilase B; 45 K = ovalbumin; 30K = carbonic anydrase; 20 K = trypsinogen and15 K = beta-lactoglobulin, we could detect thepresence of several components gp85, gp35,p27, p19, p15, p12 and probably a reversetranscriptase line in the 90 K region. The clec-trophoresis profiles showed that the layer at35% contained the complete virus or intact virusparticles.

The table 1, presents the protein content ofthe virus samples obtained after sucrose gradientcentrifugation. The viruses concentrated in themain layer were used for further processing.

Radioimmunoassay

The purified antigens p27, p19 and p15 fromexogenous ALV were sucessfully labelled with1251-Naaccording to HUNTER34 procedure, givingspecific activities between 2 to 3 x lQ4 cpm/ngprotein.

Immune serum titration - Each immune serumwas titrated in a system containing 0.02 ml 1251_p(40 - 50,000 cpm); 0.02 ml Anti-p in serial dilu-

TABLEl

Sucrose gradient virus purifícations:protein determination

VIRUS SAMPlE' 1UTALPROTEINb

mg

Endogenous Crude extract 4.501515 Upper layer 2.00

Lower layer 1.94

Endogenous Crude extract 9.007.2 Upper layer 1.30

Intermedíate layer 3.84Lower layer 2.76

Avian leukosis Crude extract 4.50Vírus (exogenous) Vírus layer 4.00

(a) Virus purification carried out by sucrose gra-dient ultracentrifugation at 110,OOOg/90min/l0°C of the virus suspension.

(b) Protein determination according to LOWRYet alii 38 refered to in Methods.

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SOUZA-FELIPPE, J. M. M. & HIGUCm, T. - Expression of avian leukosis virus proteins p27 and pl9 in uninfect-ed chicken, drake and quail cells. Rev. Inst. Adolfo Lutz; 49(2):169-178, 1989.

tions; 0.03 ml NRS (dil.1:3) TNE buffer to com-plete the volume to 0.250 ml; a first incubation37°C/3hr was folIowed by an overnight incuba-tion at 4°C; the addition of 0.03 ml GAR or SAR(dil. 1:36) and then a precipitation to comple-tion at 4°C/overnight. The pellet was collectedby centrifugation at 3,500 xg/20min., washedtwice and the radioactivity was measured in thegamma counter. Under the experimental condi-tions used, the following titre were obtained:Anti-p27 = 1:2,000, Anti-pl9 = 1:4,000 andAnti-pl5 = 1:5,000 (table not shown).

The standard competition curve was used todetermine the concentration of the equivalents ofantigen present in the system. It was preparedby using as standard competitor unlabelled puri-fied antigen in several concentration. It was al-lowed to compete with fixed amounts of labelIedantigen, in the combination with the antibodypresent in limited concentration under definedexperimental conditions. The figure 2 shows thestandard curves of antigens p27, p19 and p l S inthe system 125I_pvs Anti-p respectively.

I~

I-••••

/-123 4567

PICTURE 1 - Polyacrylamide sodium dodecyl sulphategel electrophoresis (5-20% gradient) of virus extracts.Lanes 1: Endogenous 1515-sucrose gradient upperlayer; 2: E-1515 - lower layer; 3: E-1515 - supematant;4: Endogenous 7.2 - upper layer; 5: Endogenous 7.2 -lower layer; 6 and 7: Molecular weight standards.

172

The purification of proteins from exogenousvirus particles was carried out Sepharose 6B-satured with 6 M guanidine - hydrochloride undervery special conditions (see Methods). The com-ponents were nicely separated exactly as shownby GREEN & BOLOGNESI18. Unfortunatel1y, thepurification of endogenous virus componentscould not be done in the same experimental con-ditions, since the behaviour is not alike, givingan indication of .the difference in the propertiesof the components. Instead of giving distinctpeaks in the Sepharose column as in the case ofexogenous virus, the collected fractions from en-dogenous contained all three major antigens, de-tected by radioimmunoassay. As shown in the

TABLE2

Protein eoneentration of fraetions eluted fromSepharose 6B-GuHCL eolumn

SAMPLE'

8.000.934.681.72

TOTALPRarEINb

mg

Crude extractFraction IFraction IIFraction III

(a) 9.0 mg of vírus extract disrupted and applied toSepharose 6B-GuHCL eolumn (1.4x 100 em) elut-ed with 20 mM sodium phosphate buffer ph 6.5eontaining 0.1% beta-mereaptoethanol and 6M-GuHCL.

(b) Protein content evaluated by BRADFORDmethod as refered to in Methods.

100

"'"80

E"s..'u"•..""c,

40f<l'

P15P19P27

2 4 10ng - competitor

30

PICTURE 2 - Standard competition radioimmunoassay:Assay carried out according to description in Materialsand Methods. 125I_p vs Anti-p in dilution equivalent toserum titre vs unlabelled protein, amount indicated inthe Figure; precipitation with second antibody and col-lected pellet radioactivity measured in gamma counter.

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avian leukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev. Inst, Adolfo Lutz; 49(2):169-178, 1989.

Fraction I Fraction TI

100 - Jl P19PIS

PIS

60 - P27

"eooS"'6,.9~~

P19

30 -

10 Ilg - protein 10

PICTURE 3 - Detection of components in the fractions I and II eluted from Sepharose 6B-GuHCl column: Competi-tion assay according to procedure described in "Methods". 125I_p vs Anti-p vs different amount of fraction I and TI;precipitation with second antibody; radioactivity of the pellets determined in gamma counter.

TABLE3

Competition assay: 125r-p27vs Anti-27 vs chickenembryo fibroblasts

Competitor" Precipitation" BlockagC

f.lg % %

10 98.9 1.130 88.7 11.360 76.4 23.680 72.2 27.8180 54.4 45.6

(a) Chicken embryo fobroblasts suspension sonicatedin the presence of TRITON X-I00 0.2% in thepresence of lmM PMSF. Suspension centrifugedat 1,500 xg/20min/4°e. Further centrifugations at10,000 xg/60min/4°C. The supernatant was usedas "extract",

(b) The competiton assay was carried out in a systemcontaining 0.03 ml NRS di! 1:3; 0.02 ml Anti-p27 di! 1:2,000; 0.02 mI125I_p27; competitor inthe indicated concentration, TNE . 2 to volume0.25 ml and 0.03 ml GAR dil 1:250. Pellet col-lected by centrifugation and the amount of radiac-tivity of the pellet measured in gamma counter.100% precipitation is the cprn obtainned in atube without any competitor.

(c) The percentage of blockage is calculated by sub-tracting from 100 the percentage of precitation inthe columnb•

figure 3, both fractions contained similaramounts of p27 and p15 although the p19 waspresent mainly in the first fraction. The distribu-tion isindicative of the difficulties of havingthe antigens purified by Sepharose 6B-guanidine- hydrochloride procedure, for sure more carefulstudies of the nature of the endogenous virusproteins should be done.

The expression of the equivalents to epitopesof antigens p27, p15 and p19 in chicken em-bryo fibroblasts were measured in the systemanalogous to the standard competition assay ex-cept in this case the competitor was the extractof chicken embryo fibroblasts. We found veryhigh levei of analogous to p27 and also expres-sion of components analogous to p15, indicat-ing the presence of endogenous provirus in theso called normal cells. As shown in the table 3,30 J.Lgof chicken embryo fibroblasts blocked theprecipitation reaction in about 11,3%, what isequivalent to 5 ng of p27. On the other hand, 40J.Lgcontained about 3 ng of p15.

Several authors,,6,1',39 had shown the expres-sion of certain viral genomic region in the em-bryogenesis but their activity scems to be re-pressed in the adulthood. There are diffcrence in

173

SOUZA-FELIPPE,J. M. M. & HIGUCHI, T. - Expression of avian leukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev . Inst. Adolfo Lutz; 49(2):169-178, 1989.

TABLE4 DISCUSSION

Cmpetition of normal cells from chicken, quailand drake in the system 125I_pAnti-p

Competitorng of equivalentsp19 p27

Brain Chicken 5.0 17.5Quail 36.8 33.08Drake 32.3

Heart Chicken 4.8 10.0Quail zero zeroDrake 58.8 44.3

Wingmuscle Chicken 2.1 9.3Quail 24.5 8.3Drake 39.5 8.7

Tight muscle Chicken 3.0 4.0Quail 46.5 10.5Drake 29.9

SYSTEM:Cellular or tissue extracts in severalprotein concentration: 0.06 ml NRSdil. 1:3; 0.02 rn1 of 125I_p(40-45,000cpm); 3rC/3h; 4°C overnight; 0.06ml SAR: 4°C/overnight; collected andcounted. Table calculated by normaliz-ing all competitor protein concentra-tion to 5.0 mg in order to compare thelevei of competition.

the levei of repression depending on the tissuedifferentiation Ievel. In our experiments therewere a variation of equivalent to viral antigens,depending not only on the tissues but also onthe source of chicken tested, giving a clue of theimportance of the enviromental conditions andthe species of bird used. Very little variation inthe amount of p27 and p15 was found in tighmuscle using competitor in the range of 75 to300 mg. On the other hand, brain and heart ex-tracts showed considerable levei of p27 (table4). The reaction was highly specific, there wereno inespecific precipitations, in all assays indi-vidual controls were carried out.

Cellular extracts from quails were tested in asystem similar to chicken cells, in other words,brain, heart, tigh and wing muscles were used ascompetitor. Higher expression of p27 analogouswas found in tigh muscle and then wing musele,but no competition was found in heart and braintissues (table 4).

When drake cells extracts were used, there wereno significant variation in the levei of p19 inbrain, heart and wing musele. But, the heart mus-ele showed the blockage of 28.73% when 394.4ug of tissues was used in the system 125I_p27 vsAnti-p27 vs heart musele extract (table 4).

174

Several attempts have been made in order toset the viral andogenous structural componentsof avian retrovirus in the purified form, withoutmuch success. GREEN & BOLOGNESI18 improvedthe separation procedure for viral exogenous an-tigens by using agarose gel column in the pres-ence of 6M-guanidine hydrochloride. This ap-proach was used previously in order to determinethe protein molecular weight through a simplerelationship of chain lenght and elution volumein the solvent.

We used Sepharose 6B column satured with 6M-Gu-HCl, which made the gel system very vis-cous, being so, it was necessary to adjust theflow rate in order to get adequate separation. Theprotein diffusion coefficient in 6 M-GuHCI isusually very low, elos e to the native protein.The protein renaturation was achieved by dialy-sis against a solution containing beta-mercaptohetanol and EDT A (see Methods). Theviral exogenous antigens were nicely separatedby this procedure (see Results).

In order to standardized the abbreviations usedin the field AUGUST et a1.4 suggested a standardnomenelature for vírus components: p for pro-tein; gp for glycoprotein; pp for phosphopro-tein and Pr for precursors because of the diversi-ty of abbreviations by several authors.

HIGUCHI26, HIGUCHI & AUGUST27,28tried tocorrelate the appearance of detectable group spe-cific antigen and cellular transformation, surpris-ingly at that time they were simultaneous, theappearance of group specific antigen seemed tobe essential for exogenous virus replication butnot enough to start cell transformation. The im-portance of p27 of avian system and p30 of mu-rine virus was extensively studied by Higuchiê"and HIGUCHI & AUGUSP7,28.

Several groups of investigators8,21,26,27.28,30,43,49,51,52developed the radioimmunoassay in order to de-tect minimal amount of viral antigen or itsequivalent in a system, and also to investigatethe expression of viral genes present in the hostcell,

We used the compeunon radioimmunoassayin order to detect the expression of antigens orits equivalents. We investigated the expressionand the levei of the presence of equivalents top27 and p19 in several tissues and organs ofadult chickens as well as in chicken embryo fib-roblasts. Since the ability to detect them de-pends on the amount of competitor used no com-petition was found at protein concentration inthe range of 0.1 to 0.2 mg. However, when the

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avian leukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev . Inst. Adolfo Lutz, 49(2):169~178, 1989.

amount of competitor, in the experimental con-ditions was increased, epitopes of p27 and p19were detected!? as shown in table 4 and also ofp15 (date no shown).

When the system 125I_p27vs Anti-p27 vs cel-lular extracts were used, 3.85 mg of chickenbrain material blocked 50% the precipitation re-action, it was equivalent to 28 ng of p27. Onthe other hand, almost twice as much 5.52 mg ofheart extract was necessary to give the same in-hibition. Surprisingly, tigh muscle and wingmuscle did not shown significant competitionfor any of labelled antigens.

The p19 is associated with genomic RNA andis linked to lipids and the outer core. Sometimesit is phosphorilated. It might be important forRNA packaging'6.37.45,50.

A different pattern of expression was foundfor p19 expression; all sample analysed showedcompetition, which might be due to some simi-larity between the p19 and some cellular cornpo-nents, although the control values were takeninto account.

When the same tissues and organs extractsfrom quail and drake were analysed, similar com-petition pattern were found, although at differentprotein concentrations. The experimental resultssuggest more similarity between the endogenousproteins of chicken and drake than with the quailproteins. Moreover, the radioimmunoassay re-sults showed satisfactory immunological reactionwith the exogenous virus antigens p27, p19 andp15 and the endogenous components, whichstrenghens the assumption that the gag genesare conserved among avian retrovirus.

Making several crosses among white Leghornchickens, CRITTENDEN'4 isolated cell strains,5.7 and 17 carrying a dominant gene predispos-ing the cell to spontaneous activation of endog-enous viruses genomes. The line 7.2 is hornozy-gous for the gene V-E7 and lacks the dominanthost gene Gs and H-E. The line 15 is inducible itproduces non infectious particles by BrdU treat-rnenr" and it is an unique line.

MARTIN et a1.39 reported the recovery of en-dogenous retrovirus from baboon, stump-tail ma-caque and colobus, but, so far, endogenous retro-viral DNA has not been detected in preparationswith normal human DNA probes.

There are considerable amount of speculationsabout the mechanisms used for integrative rec-ombination of retroviral DNA in the cellularchromosome. Surely, there must be an endoge-nous control, that could be broken by one orcombination of immunological, hormonal, en-viromental factors; mutagenic elements'v-" pro-motion of transcription of cellular DNA41.44,46,on-co genesv-" and transpossonsr-»-». It seems to beparticularly important the region of insertion be-cause it might promote or activate the full ex-pression of relevant genes.

Acknowledgement

We would like to express our gratitude toProf. Dr. A.A. Pupo (Medical School - USP) forhis kindness in allowing us to use the gammacounter. The culture fluid was kindly supplied byDr. C.H. Romero (EMBRAP A). The laboratorywas set up because of the FAPESP grants ns. 81/0215-4, 82/1592-9 and 84/0384-9.

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SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expression of avian leukosis virus proteins p27 and p19 in uninfect-ed chicken, drake and quail cells. Rev. Inst. Adolfo Lutz; 49(2):169-178, 1989.

RIALA6/675

SOUZA-FELIPPE, J. M. M. & HIGUCHI, T. - Expressão das proteínas p27 e p19 devírus de leucose aviária em células não infectadas de galinha, pato e marreco.Rev. Inst. Adolfo Lutz, 49(2):169-178, 1989.

RESUMO: Células animais normais, não infectadas, apresentam uma característicaúnica; a presença em seu genoma de genes (provirus) idênticos ou estritamenterelacionados a retrovirus exógenos infecciosos. O nível de expressão destes genesvaria de indivíduo para indivíduo: do silêncio completo até a produção de partículasvirais, Proteínas de vírus endógenos e exógenos foram purificadas a partir de vírussonicados e passados em coluna de Sepharose 6B saturada com GuHCI 6M. A presençade equivalentes dos antígenos p27 e p 19 foi analisada por radioimunoensaio. Apresença de componentes de vírus endógenos foi investigada em CEF, coração,cérebro, músculo de asa e músculo de coxa de galinha, codorna e marreco. A função ousignificado destas proteínas é desconhecida. Fatores ambientais, hormonais eimunológicos necessitam ser estudados.

DESCRITORES: Retrovírus; vírus endógeno; expressão de vírus endógenos; RIA.

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Recebido para publicação em 17 de maio de 1989.