Dr. ?aul mugb
CQld Spring P.hr Laberatmy of

Cold Spring Earber

Lmg Ilbnd, ROW York

Dear Dr. ftough:

My colloapo Xike Birhop ra  ntly roturn-d fwm c8H with a report (via mncy

Eopkinr) that yeu have boon extrawly ruccorrful In enuclutfng relatively large
numbotr ef collr and that you might bo lntarorted in a collabsrative venture
de811ng with the machanim +f previral Dmu ryntheria by Eaur rarcoma virur.
the accrganying pmprintr indicator, uo have bean 8tudgIn.ag the rynthorir of RSV-
rpacific EUA in both polwiatire (duck) and non-pemirrive (-fisn, mainly murine)

Quantitative Bfolegy

As

ho6tS.

In duck cells we can dotect viral DMA rynthoris a8 early 8s 3 hours after infectim
(by rubgroup C virurer), and at thir point all of tho DRSA ir in the cyteplarm a8
detormined by assay of convontionallyrfractionated cympl8m and nucleic.
uro Waahed, rwollea in 0.1x8811, bunced in the prerence of 1)40 and Doc, and nucloi
rpua out at lev rpeed.)  The smmunt of DiU increaror in the whola cell up to 16-24
hours, but by 16 hours it ir dotoctable only in the nuclear fraction.  Integrstien
(or measured by tho nohrark teat) begin8 by 9 hsurr or se, and is coapleted within
24-36 hours.  ?roliminary risln$ af olthor cytoplawaic ar unintegratod DNA euggertr
that the in vlve product ir about the
a double atrandad melocule of about 6x106.  Early in infoction, thore appear8 t0 be
both ringle snd deuble stranded DRIS in tho cytsplanr.
DMA is infoctious, but we are now testing this.  We alto ah0 in the precera 0f
lsaking at the rurcopt~bllity of unintogratrd (and integrated) proviral D#A to re-
striction onbnuclaaras.

(Colla

of 8 copy of a subunit of R#b, f.0.9

We do not yet know if thir

Viral llloA ryntherir by RSV aftor mars infection of maaPurlian cell8 (we u8e Todaro'r
A31 clono of BALB/c 3T3 cell.) ir @b8erpab1% only with virus previously mrcued(by
cocultiv8tion with a pewisrive bot) frsa UV-transfon#ld mouma er rat cella.
?rerumably thlr phenomenen is related to the increared e ficiency of transformatiam
(from vory poor to poor--4.e. , fmm 10-5 to 10.4 or loof in ccplpariton with focus
formation in chick cells) by rercuod virur, a8 noted by Altanrar and Tarin.  DNA
ryntheair (to the extont of 1-2 cogier/eall) is detectable by 12 hours (and porhapr
earlier---we haven't lookod); integration begiar uftor 12 hourr and io complete
within 60 hours; the PBlA L8 atably Lnbortted fer at least revera1 generatlbnr.  It
Is overplholmingly likoly that the vast majority ef cells aro infocted (80 warured
by viral DNA ryntheris) but not tranrformad (as meaaurad by ability to clrpne in
@oft agar); we are in the precess erf terting thir mer. rtringontly by examining clenos
of infected colla knmm net to grew in soft agar.
largely absrtiwe infclctlon thero is considerablo trunrcription of viral

$urprisingly, 60 hour8 after thir
               (as much

Dr. tau1 Haurgh
Augurt 16, 1973

?ago 2

as 10 tims more lulpA than we roo in a clone of tranrfonaod meu.8 celb 8nd it ir
copkd froe at lort 80% of tho gelsaw a0 warured by a coa).titLoa hybridisotisa

8808y) o

f hope you will agree with me that a furion of our technelogior might allow ua to
ark 8- vary intererting quertionr abut the ryntherir and uharactorirticr of
prsviral DRa in there torr, types of hort cell.

ryntherir of 1-4 copier af viral DLU per cell.
aultiplicitier of infecticm and pmlmged incubation slight result in mom viral
IRU syntherir par cell with onucloated cella.  Uuu miany cello can mu pmcosr at
onae (tho report I heard waa lO7)? What varieties ef cellr have you tried? Haw
long do your cellr min healthy after enuclutiaa?  What parcoat -in nucleated
(rumor ha0 it thir ir lera than lX)?

To give you 001. idea of tho

10gfOtfCB that might b@ nguirold, YI Cumt1y We about 5-10~107 Cell0 to 1I8IUrtl

It fr caaceivable that higbor

If yeu ero intererted in purruing 1010 of there matterr, WT could proceed tn a
number ef wyr: I oould rinqly rond you tho necesa8ry virurar and receive the
p8rti811y axtrected cello back from you; if convenient for YOU, you wauld be
uelcomo to ctma out and work in our lab for a couplo of weeks to get thir offart
started; cenceivably, I could eem there for a short period, equippod with vlnrrer,
but a rm-to-be-delivered firrt b8by maker this prerpoet unlikely in the noar
future.  ??&me mito or call ma here (415-666-2824) when pou've had ar chance to
digest this, and wa can conridor tho perribilitier further.

Yourr ,

Harold E. Vararur, H.D.
Arsiatant Irroferror
Dapartwut of Wcrobiologp

Itncl: EBV: Lepotit

sqyw va11.y

?HAS - Intogration papor
J. Wl. Biol. reprint