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PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines
Life Sciences, Vol. 50, pp. 1973-1984 Printed in the USA
PCR
AMPLIFICATION OF LARGE GENOMIC FRAGMENTS IMMUNODEFICIENCY VIRUS INFECTED
Nitin Paul
Pergamon Press
FROM CELL
HUMAN LINES
AND
SIMIAN
K. S a k s e n a * @ , D o m i n i c E. Dwyer***, Michaela M~ller**, Durand****, Vincent Herv~****, Marie-Claude Lang** Fran~oise Barr~-Sinoussi.**
Jean and
* S U N Y , H S C . D e p a r t m e n t of M e d i c i n e , R o o m 1 3 0 5 , W S K , S y r a c u s e , N . Y . 1 3 2 0 3 . * * I n s t i t u t e Pasteur, Biologie des Retrovirus, 25 Rue du Dr. Roux, Paris France. * * * I n s t i t u t e for Clinical pathology and Medical Research, W e s t m e a d Hospital, W e s t m e a d N3W 2145, Australia. * * * * I n s t i t u t e Pasteur, Bangui, Central African Republic. (Received in final form April i0, 1992) Snmmary
P o l y m e r a s e chain reaction (PCR) has been used to a m p l i f y the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from W e s t e r n Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from h i g h l y conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have a m p l i f i e d by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from i2.3 kb from gag pol and env genes have been s u c c e s s f u l l y amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a h i g h l y conserved stretch of the viral genome. Since the onset of PCR as a tool for amplifying DNA, various c o n d i t i o n s and parameters have been defined for its o p t i m i z a t i o n (i). Now DNA can be amplified from a fixed biological material (2). C o n s i d e r a b l e progress has been made in m o l e c u l a r biology using PCR as a technique for studying various genes from diverse p r o c a r y o t e s and eucaryotes. Presently, PCR is being used r o u t i n e l y to study human and animal retroviruses p a r t i c u l a r l y their genomic variability. This has become the most dependable t e c h n i q u e because of its high efficiency and sensitivity, provided appropriate p r e c a u t i o n s are taken to avoid false positive reactions (3, 4). Ever since the discovery of the human i m m u n o d e f i c i e n c y virus 1 and 2 (HIV-I and HIV-2) as causative agents of AIDS (5, 6)
@ A u t h o r to whom correspondence should be addressed 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
type
1974
PCR Amplification of Large Genomic Fragments
Vol. 50, No. 25, 1992
considerable progress has been made in identifying and characterizing AIDS like viruses (SIV) from various nonhuman primates (7-11). Simian immunodeficiency viruses are primate lentiviruses that share a common ancestry with human immundeficiency viruses (HIV-I and HIV-2) (12-16) and thus it has become important to study more simian i m m u n o d e f i c i e n c y viruses for b e t t e r u n d e r s t a n d i n g of the biological, g e n e t i c and also the phylogentic relationships they share with the Human i m m u n o d e f i c i e n c y virus type-i and type-2. The H I V and SIV may have low copy number in infected cells and to clone the entire viral genome becomes a tedious task and exorbitant in terms of cost. Amplifying short length genomic fragments by PCR is also inefficient and not cost effective. C o n s i d e r i n g these facts, amplifying large genomic f r a g m e n t s w o u l d not only p r o v i d e complete genetic information harbored in the viral genomes but will be less time c o n s u m i n g and m o r e cost effective and would m i n i m i z e problems of PCR c o n t a m i n a t i o n by reducing the number of reactions necessary to o b t a i n genomic m a t e r i a l for c l o n i n g and sequencing. Recent reports (17) have described the a m p l i f i c a t i o n of 5-9 kb gene f r a g m e n t s of B - C a e s i n gene cloned in a plasmid. C r e a t i o n of an i n f e c t i o u s clone of SIVsmm by PCR has also been d e s c r i b e d (18). There are no reports on the optimization of amplification conditions necessary for successfully obtaining large genomic f r a g m e n t s from a retrovirus (HIV and SIV) infected stock. In the p r e s e n t investigation we have defined and o p t i m i z e d the c o n d i t i o n s for the a m p l i f i c a t i o n of large g e n o m i c f r a g m e n t s (i5.5 kb) d i r e c t l y from the HIV2 and SIV infected Molt4 clone-8 cell line, and not from the genes cloned into plasmids. We describe the p o t e n t i a l of PCR in extracting more genetic i n f o r m a t i o n from these p a t h o g e i c viruses in a shorter time and cost e f f e c t i v e manner.
Materials
and Methods
Animals: The A f r i c a n monkeys (Erythrocebus) used in this study were w i l d caught in W e s t e r n Africa in Senegal at the I n s t i t u t e Pasteur. All the animals were healthy and asymptomatic. P e r i p h e r a l blood I y m p h o c y t e s (PBL) from monkeys were sampled and c u l t u r e d w i t h Molt-4 clone 8 cells. Two asymptomatic, seronegative adult females (89005 and 89008 sampled for PBL on 4.26.1989) w e r e used for t h e s e observations. Cell lines and virus culture: PBLs were isolated on p e r c o l l and co-cultivated with C8166 cells in the presence of phytohemagglutinin (PHA). Subsequently, Molt-4 clone 8 cells were used as target cells for sir infection. Culture was done in RPMI 1640 medium supplemented with i00 IU/ml penicillin, 100ug/ml streptomycin and 10% fetal calf serum (FCS). Cultures were maintained at 37°C in a 5% CO 2 incubator and the m e d i u m was r e n e w e d twice a week. The appearance of syncytia s u g g e s t e d the p r e s e n c e of SIV and cytopathic effects (CPE) were o b s e r v e d under light microscopy. Also for HIV-2 (ROD) we used Molt-4 clone 8 cells w h i c h were cultured and h a r v e s t e d after two p a s s a g e s when c y t o p a t h i c effects were visible under light microscopy. U n i n f e c t e d
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
Molt-4 clone 8 cells were maintained parallel as a negative control.
in
continuous
1975
culture
in
Reverse Transcriptase (RT) activity: We measured the DNA p o l y m e r a s e activity [poly (A) dependent] in 50ul of 50mM tris hydrochloride (pH7.5), 5mM dithiothreitol, 100mM KCI, 10mM M a g n e s i u m chloride, 10mM[3H]dTTP, 0.1% Triton X-100 c o n t a i n i n g 2ul of poly (A), 0.4ug of oligo (dT), and an a p p r o p r i a t e amount of virions. The reaction was carried out at 37°C for 1 hour. The polymerized [3H]dTTP was precipitated with 10% t r i c h l o r o a c e t i c acid, c o l l e c t e d on a membrane filter (Millipore Corp., Bedford, Mass), w a s h e d with 10% trichloroacetic acid and ethanol, air dried, and counted in a liquid scintillation counter (5). Electron Microscopy: Virus infected Molt-4 clone 8 cells were fixed in g l u t a c a c o d y l a t e and osmium tetraoxide, and then embedded in araldite. These sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope. Extraction and analysis of uninteqrated viral DNA: U n i n t e g r a t e d HIV-2 (ROD) and SIV DNA was extracted following the m e t h o d of Hirt (15). Briefly, aliquots of 4x106 cells were lysed at 65°C in 0.6% sodium dodecyl sulfate (SDS). Hirt supernatants were e x t r a c t e d twice with phonel, once chloroform and p r e c i p i t a t e d with ethanol. All the cell cultures and the DNA extractions for HIV-2 and SIV were c a r r i e d out under separate P2 biosafety hoods in order to avoid carry over problems. The DNA samples were a l i q u o t e d under the UV s t e r i l i z e d biosafety hoods. Before p r o c e e d i n g for PCR, some of the DNA extracts were electrophoresed in 0.8% tris borate (pH 8.0) agarose gels and transferred to nylon m e m b r a n e filters (Amersham International, UK). Filters were p r e h y b r i d i z e d at 42°C in a solution containing 5xSSC (lxSSC is 0.015M sodium citrate plus o.15M NaCl), 50% formamide, 1% SDS, 3x Denhardt solution, 100ug of d e n a t u r e d salmon sperm DNA/ml and SuM EDTA (pH 8.0). H y b r i d i z a t i o n was done overnight with 2x106 cpm of [alpha-32p] labelled DNA probe per ml (specificity activity, 2x108 to 5xl0Ucpm/ug), and the filters were washed under a g i t a t i o n in 0.2% SSC, 0.1% SDS at 60-65°C. Washed filters were blotted dry, w r a p p e d in saran wrap and subjected to a u t o r a d i o g r a p h y with X-omat i n t e n s i f y i n g screens (Eastman Kodak Co., Rochester, N.Y.) (19). Standard polymerase chain reaction: PCR were p e r f o r m e d in 500ul Eppendorf tubes in a total volume of i00 ul c o n t a i n i n g the following: 10mM Tris HCI (pH 8.3), 50 mM KCI, 1.5 mM MgC12, 0.01% w/v gelatin, 200 mM each of dATP, dCTP, dGTP, dTTP, 2mM of each primer, lug of DNA template, 3U of taq p o l y m e r a s e (Cetus corp, Norwalk, CT). Reactions were overlaid with 60ul of mineral oil and a m p l i f i e d for 35 cycles using an automated t e m p e r a t u r e cycler (LEP Thermocycler, UK). We changed the extension time to f a c i l i t a t e the a m p l i f i c a t i o n of fragments of different sizes. Specific primer sequences and their genomic locations are listed in Table II. i) Amplifyinq ikb-2kb fraqments: 95°C for 5 min., 55°c for 1 min. and 72°C for 2 min. 95°C for 30 sec., 55°C for 1 min. and 72°C for 2 min. 95°C for 30 sec., 55°C for 1 min. and 72°C for 7 min.
(i cycle) (33 cycles) (i cycle)
1976
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
2) Amplifyinq fraqments > 3kb: 9 5 ° C for 5 min., 5 5 ° c for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min. 9 5 ° C f o r 30 sec., 55°C for 1 min.
and 72°C and 7 2 ° C and 7 2 ° C
for for for
4 min. 4 min. i0 min.
3) Amplifyinq fraqments > 4kb: 9 5 ° C for 5 min., 5 5 ° c for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min.
and 72°C and 72°C and 72°C
for for for
6 min. (cycle) 6 m i n (33 cycles) i0 m i n (33 cycles)
(i cycle) (35 cycles) (i cycle)
A n a l y s i s of PCR products: T w e n t y m i c r o l i t e r s of P C R a m p l i f i e d p r o d u c t w e r e a n a l y z e d on 0.8% a g a r o s e gel. A f t e r e l e c t r o p h o r e s i s for 1 h o u r at 150V, the D N A w a s v i e w e d u n d e r U V t r a n s i l l u m i n a t o r . D N A w a s t r a n s f e r r e d on t h e N y l o n m e m b r a n e a n d h y b r i d i z e d a c c o r d i n g t o t h e p r o t o c o l as d e s c i b e d above. Sequence
SI.No. 1 2 3 4 7 8 9 i0 ii 12 13 14 15 16 17 18 19 20 The
Primer
Table I of o l i g o n u c l e o t i d e P r i m e r s
Location
Sequence
5'-
Used
- - 3'
P2a P2b P7a P7b
env env env env
GCCTGAATAATTGGTATCATTACA TGTAATGATACCAATTATTCAGGC CATTTCCTGATCCGCCAGCTGAT ATCAGCTGGCGGATCAGGAAATG
JFPI JFP2 JFP3a JFP3b JFP4 JFGI
pol pol pol plo pol gag
CAAGCTTAGGAATACCACACCC CAAGCTTGATATACAGAAGTTAGT
JFG2 gag JFEI env JFE2 env D D 916 gag-pol D D 939 gag-pol D D 6512 gag-pol DDI0169 env-ltr DDI0269 env-ltr sequence before the
in P C R
AAAGCTT/GGGTACCAGCACACAAAGG AAAGCTT/TGTGTGCTGGTACCCA
AAAGCTT/GCTCCTTCCCTTTCCA
Consensus between SIVmaC/ HIV-2
HIV-2 SIVsm SIVmac SIVagm TYO-I
AGAATTCCGCCCGAACAGGGACTTGA TGAATTCAGGCCGTCAGCATTTCTTC CGAATTC/AAAAAGTGTTGCTACCA
CGAATTC/CAATAATCTTTGTCACA GGCAGTAAGGGCGGCAGGAAC AACCACGACGGAGTGCTCCTA GGAAATCCTCTCTCAGCTATACCG TGAATTGTGCCCAGCACCGGCCAAGTG ATCTCTCCTAGTCGCCGCCTGGTGAC s l a s h (/) i n d i c a t e s t h e l i n k e r
HIV-2/ SIVmac HIV-2 HIV-2 HIV-2
ROD NIH-2 ISY
sequence
Results E l e c t r o n microscopy: T h e p r e s e n c e of v i r u s in t h e t w o i n f e c t e d m o n k e y s , 8 9 0 0 5 a n d 8 9 0 0 8 (fig. IA a n d IB) w a s c o n f i r m e d i n i t i a l l y by e l e c t r o n m i c r o s c o p y . M o l t - 4 a n d c l o n e 8 c e l l s r e v e a l e d H I V / S I V like retroviral particles. Viral particles, after 6 days of culture, c o u l d be s e e n b u d d i n g f r o m t h e c e l l s u r f a c e in c a s e of 8 9 0 0 5 i n f e c t e d c e l l s (fig. IA) and s e v e r e C P E w i t h m a s s i v e c e l l death and high viral load w a s s e e n in c a s e of 8 9 0 0 8 infected cells, a l s o a f t e r 6 d a y s of c u l t u r e (fig. IB). T h i s i n d i c a t e d t h a t the E r y t h r o c e b u s m o n k e y s h a r b o r e d SIV in t h e i r w i d h a b i t a t . U n d e r light microscopy large syncytia and cell death with large c e l l u l a r s p a c e s w e r e a l s o o b s e r v e d ( r e s u l t s n o t shown).
Vol. 50, No. 25, 1992
PCR Amplification
Viral Consensus Sequence Between Code HIV-2 SIVagm SIVmac SIVsmm
T a b l e II of D N A f r o m I s o l a t e s 89005, SIVmac
Genome
Region
JFG JFPl-3b JFPI-4 JFP2-3b JFP2-4 JFP3a-4
HIV-2 SIVmac HIV-2 (ROD)
PCR Amplification of Large Genomic Fragments
JF E P2b-P7b
DD 916-6442
HIV-2
Amplification
Position
Fragment Length(bp)
gag pol pol pol pol pol
315-1600 2610-3960 2610-4765 3100-3960 3100-4765 3935-4765
1285 1350 2155 860 1665 830
env env
6230-8430 6825-8430
800 1600
gag-pol
916-6442
HIV-2 NIH-2 HIV-2 ISY
89008,
5200
1977
(ROD)
and
Products
89005
89008
HIV-2
SImac
-* + + + + +
-NS +NS +NS +NS +NS +NS
-* + + + + +
-* + + +
+
+
+
NT
+
+
+
NT
5500 DD65212-I0171
env-LTR
6512-10171
3500 3700
H y b r i d i z a t i o n w a s d o n e w i t h H I V - 2 (ROD) e n t i r e probe (gag-pol-env-ltr) under high stringency conditions and with stringent washes
NS
- Not
Shown
NT - N o t T e s t e d
* T e s t e d n e g a t i v e b e c a u s e H I V 2 (ROD) p r o b e w a s l a c k i n g g a g r e g i o n (315-1600) + - Amplification and Hybridization positive
Reverse
Transcriptase activity: A f t e r 6 d a y s of c u l t u r e of 8 9 0 0 5 and 89008 infected Molt-4 clone 8 cells showed low t i t e r s of r e v e r s e R T a c t i v i t y in t h e c u l t u r e s u p e r n a t a n t s . In c o n t r a s t , t h e S I V m a c a n d H I V - 2 (ROD) i n f e c t e d M o l t - 4 c l o n e 8 c e l l s s h o w e d h i g h t i t e r s of R T a c t i v i t y at the end of s e c o n d p a s s a g e ( r e s u l t s n o t shown)
Southern blot hybridization: Before PCR amplification, the Hirt DNA preparation from infected cells was hybridized with SIVmac (R9) p r o b e c o n t a i n i n g t h e e n t i r e v i r a l g e n o m e . T h e D N A f r o m M o l t - 4 c l o n e 8 c e l l s i n f e c t e d w i t h S I V i s o l a t e s (89005 a n d 89008) s h o w e d hybridization s i g n a l s , t h e r e b y i n d i c a t i n g t h e p r e s e n c e of v i r a l DNA. T h e s e o b s e r v a t i o n s c o n c u r r e d w i t h the r e s u l t s o b t a i n e d w i t h electron microscopy. The DNA from the isolate 89008 also showed t h e p r e s e n c e of u n i n t e g r a t e d v i r a l D N A of a b o u t 9 k b (fig. 2). Similarly, DNA from Molt-4 clone 8 infected with SIVmac and HIV-2 (ROD) also showed good hybridization signals with SIVmac (R9) probe.
1978
PCR Amplification of Large Genomic Fragments
FIG.1A
and
Vol. 50, No. 25, 1992
1B
Electron microspy of 8 9 0 0 5 and 89008 infected Molt-4 clone 8 cells. A. SIV (89005) infected Molt-4 clone 8 cells showing b u d d i n g of v i r u s p a r t i c l e f r o m t h e c e l l s u r f a c e a f t e r 6 days of i n f e c t i o n . M a g x l 6 0 0 . 0 0 0 . B. S I V (89008) i n f e c t e d Molt-4 clone 8 cells showing mature virus particles after 6 days of i n f e c t i o n . M a g x 200,000. v i r u s p a r t i c l e s c a n be s e e n i n s i d e t h e d e a d cells. (Arrows indicate the budding and the mature virus particles.)
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
1979
05
1 A I
C D
E
DNA
FIG.
2
Southern blot hybridization H i r t p r e p a r a t i o n of viral DNA f r o m M o l t - 4 c l o n e 8 c e l l s infected with 89005, 89008, SIVmac and HIV-2 (ROD). S I V m a c (R9), c o n t a i n i n g the e n t i r e v i r a l genome, h a s b e e n u s e d as a probe. L a n e A. D N A f r o m 89005 i n f e c t e d cells. L a n e B. D N A f r o m 89008 i n f e c t e d cells. L a n e s C a n d D. D N A f r o m S I V m a c and HIV-2 (ROD) i n f e c t e d cells.
PCR amplification: We h a v e s i m p l i f i e d the P C R a m p l i f i c a t i o n of long genomic fragments from HIV-2 (ROD) a n d S I V i n f e c t e d cell lines by using consensus primer sequences taken from highly c o n s e r v e d r e g i o n s b e t w e e n S I V m a c and HIV-2. F i g u r e 3 s h o w s t h a t we have amplified D N A f r o m the 89005 isolate. The result of P C R a m p l i f i c a t i o n w i t h D N A f r o m 89008 h a v e b e e n t a b u l a t e d in t a b l e II. In o r d e r to b e t t e r o p t i m i z e the c o n d i t i o n s for a m p l i f i c a t i o n of l a r g e g e n o m i c s e g m e n t s of r e t r o v i r a l g e n e s we h a v e a s s a y e d v a r i o u s overlapping oligonucleotides extending over the entire viral g e n o m e (LTR, gag, pol,and env) (Table i).
1980
PCR Amplification of Large Genomic Fragments
A
B
C
O
E
F
O
P9.11
,,
O
H
~
J
K
Vol. 50, No. 25, 1992
L
M
N
O
el
~
.
+
.
X +1.
2
3
~e FIG • 3 P C R a m p l i f i c a t i o n of g e n o m i c f r a g m e n t s r a n g i n g f r o m 8 0 0 2 1 5 0 b p f r o m E r y t h r o c e b u s i s o l a t e (89005), HIV-2 (ROD) a n d S I V m a c by u s i n g c o n s e n s u s p r i m e r s e q u e n c e s b e t w e e n SIVmac, H I V - 2 a n d SIVsm. L A N E S A , B , C , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F G (1300bp) L A N E S D , E , F , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P l - 3 B ( 1 3 5 0 b p ) L A N E S G , H , I , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 2 - 3 B (860bp) L A N E S J , K , L , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P I - 4 (2150bp) L A N E S M , N , O , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 2 - 4 (1660bp) L A N E S P , Q , R , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 3 - 4 (8200bp) L A N E S S , T , U , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F E (800bp) L A N E S V , X , Y , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r P 2 - P 7 (1600bp) L A N E S 1 , 2 , 3 , - N e g a t i v e c o n t r o l s (No DNA: D N A f r o m n o n i n f e c t e d M o l t 4 c l o n e 8 cells, and no p r i m e r control. H y b r i d i z a t i o n was c a r r i e d out w i t h HIV-2 (ROD) probe, l a c k i n g the g a g region, u n d e r h i g h s t r i n g e n c y c o n d i t i o n s
(65°c).
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
1981
Using this strategy we have successfully a m p l i f i e d viral gene fragments ranging from 830bp-2155bp (fig.3 and Table II). The e x t e n s i o n cycle in this case was 1-3 minutes. To be more sure of amplification efficiency of fragments between 1000-2000bp we d e s i g n e d several primer pairs in gag and pol regions along with some o v e r l a p p i n g primer pairs which amplified 1285bp in gag and 1350, 860, 2155, 1665 and 830bp in pol gene. All of them were hybridization positive with HIV-2 (ROD) except the SIVmac. C o n s i d e r i n g the fact that HIV-2ROD and SIVmac are 35% d i v e r g e n t from each other, the absence of signals under high s t r i n g e n c y hybridization conditions can be expected. These results d e m o n s t r a t e that the fragments ranging from 8 0 0 - 2 1 5 5 b p may be e f f i c i e n t l y amplified by using extension time between 1-3 minutes per cycle. No h y b r i d i z a t i o n specific a m p l i f i c a t i o n was seen with primer JFE and P2-P7 (fig.3 Lanes S,T,U and V,W,X). The a m p l i f i e d p r o d u c t s were nonspecific amplicons. Similarly, gag fragments (1285bp, Lanes A,B and C; fig. 3) did not show any hybridization. This is because of the fact that the HIV-2 (ROD) probe lacked the gag region. In another set of experiments we amplified the entire viral genome of a p p r o x i m a t e l y <9kb in two halves of 3.5 and 5.2kb. One half spans from within the U3 region of 5'LTR into pol gene and an o v e r l a p p i n g fragment spans 3700bp from the pol gene into U5 region of 3'LTR (fig. 4; Table II). In each case (fig.3 and fig.4) the a m p l i f i e d fragments when hybridized with specific genomic probes revealed the specificity of PCR.
DISCUSSION
We have d e s c r i b e d a convenient way of generating long genomic DNA fragments, from cells infected with HIV-2 (ROD) and SIV, by PCR. Our data emphasizes the potential of PCR in h a r v e s t i n g more g e n e t i c information from these complex group of viruses, which have very low copy number of viral DNA, in a shorter time. The c o n d i t i o n s defined here are cost effective and highly efficient. Several approaches have been adopted for g e n e r a t i n g short genomic fragments from viral genes, but all this is very time consuming. H a v i n g a little starting substrate material for a m p l i f i c a t i o n is also a c o n s t r a i n t in the study of these retroviruses. This makes cloning of retroviruses in conventional phages more tedious and time consuming. Long range PCR can facilitate the cloning and s e q u e n c i n g of retroviral genes in a much simpler way and this we have demonstrated by amplifying the entire genome of SIV (Erythrocebus) and HIV-2 (ROD) in two halves. A similar approach has been taken with another SIV, SIVSmmPBjI4 (18). In our analyses, it is clear that the extension time is important for e f f i c i e n t amplification of large genomic fragments. We allow one m i n u t e per kilobase as more prolonged e x t e n s i o n times favor the f o r m a t i o n of nonspecific bands. The q u a n t i t y of a m p l i f i e d large fragments obtained from samples with low copy numbers of DNA may be low, but it is usually sufficient for cloning and sequencing. If the yield is low, the PCR fragments can be r e a m p l i f i e d with the same primers or with nested primers, although case must be taken to avoid contamination (3). Occasionally, p r o b l e m s are encountered with nonspecific bands or primers dimers
1982
PCR Amplification of Large Genomic Fragments
A
B
FIG.
Vol. 50, No. 25, 1992
C
4
PCR a m p l i f i c a t i o n of env fragment (3.5 kb) and g a g - p o l fragment (5.2 kb) from DNA of Erythrocebus isolate 89005 by u s i n g d i s t a n t l y located p r i m e r s conserved in HIV-2. L a n e M - M o l e c u l a r w e i g h t m a r k e r (Lambda H i n d III d i g e s t e d ) A- D N A f r o m 89005 i n f e c t e d c e l l s (Lane A - 8 9 0 0 5 D N A a n d L a n e D - H I V - 2 DNA) s h o w i n g t h e a m p l i f i c a t i o n of 3.7 kb e n v f r a g m e n t . L a n e s B and C are n e g a t i v e c o n t r o l s . B e l o w are the hybridization results. B- D N A from 89005 and H I V - 2 i n f e c t e d cells showing amplification of 5.2 k b g a g - p o l fragment (Lane- A 89005 and lane -D HIV-2 DNA). Lanes B and C are negative controls. B e l o w are the h y b r i d i z a t i o n results. C- A l s o s h o w s t h e amplification of 89005 DNA f r o m i n f e c t e d cells. H e r e t h e 3.5 kb e n v f r a g m e n t w a s a m p l i f i e d by u s i n g a n e s t e d p r i m e r (see T a b l e I a n d II). On t h e r i g h t h a n d side are the h y b r i d i z a t i o n r e s u l t s . In all the cases the HIV-2 (ROD) probe has been used for hybridization under stringent conditions.
when reamplifying PCR products to o b t a i n enough material for s e q u e n c i n g . T h i s is p r e s u m a b l y d u e to the p r e s e n c e (often n o t s e e n on t h e gels) of non specific amplified material, a n d c a n be m i n i m i z e d by p u r i f y i n g the b a n d s of i n t e r e s t f r o m gels, e n s u r i n g t h a t t h e a l i q u o t s f r o m the p r e v i o u s P C R are h i g h l y d i l u t e d , a n d by e n s u r i n g t h e n e s t e d p r i m e r s are h i g h l y s p e c i f i c . W i t h the l o n g e r e x t e n s i o n t i m e s r e q u i r e d for large f r a g m e n t s it is i m p o r t a n t not to u s e t o o m a n y c y c l e s (eg. less t h a n 25) so t h a t n o n s p e c i f i c b a n d s a n d loss of taq p o l y m e r a s e a c t i v i t y are limited. T h i s is especially important when performing nested PCR. Excessive taq polymerse (eg. m o r e than 2-4 U n i t s per reaction) is a l s o an a v o i d a b l e c a u s e of n o n s p e c i f i c bands, e s p e c i a l l y w h e n a m p l i f y i n g f r o m m a t e r i a l c o n t a i n i n g low levels of virus. T h e o p t i m a l a m o u n t of e n z y m e n e e d s to be a s c e r t a i n e d w i t h e a c h p r i m e r pair, b u t in g e n e r a l 1 u n i t or less is all t h a t is n e c e s s a r y . Primer selection is o b v i o u s l y crucial to the success of PCR. A l t h o u g h t h e g e n e t i c v a r i a t i o n and h i g h m u t a t i o n r a t e s of H I V a n d S I V a r e w e l l k n o w n (9,12,14,18) the s e l e c t i o n of p r i m e r s u s e d in
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PCR Amplification of Large Genomic Fragments
1983
these e x p e r i m e n t s in highly conserved regions allowed r e a s o n a b l y high stringency conditions to be used. In our experience, o c c a s i o n a l m i s m a t c h e s in oligonucleotide primers did not affect the a m p l i f i c a t i o n of shorter fragments (between 100-700bp), but could affect the amplification of larger fragments (>800bp). Our analyses are useful as they demonstrate the value of PCR in a m p l i f y i n g large viral genomic fragments, from low copy genomic material, for cloning and sequencing. This may prove useful not only to retrovirologist, but can also be applied to study other viruses and lower organisms. This will facilitate the u n d e r s t a n d i n g of molecular and biological c o m p l e x i t y of these organisms in a shorter time and in a cost effective m a n n e r and will also pave the way for defining new lentiviruses. Acknowledgements
NKS is thankful to CIES, Paris (France) for financial assistance. We are indebted to Drs Luc Montagnier and Francois Clavel for HIV-2 (ROD) probe. References
i.
2 3. 4. 5.
6.
7. 8. 9. i0. ii. 12. 13.
14. 15. 16. 17.
B.J.POIES, G.D.EHRLICH, B.C.BYRNE, K.WELLS, S.KWOK, J.SNINSKY,L.M. de la Maza and E.M.Peterson (eds). Medical V i r o l o g y 9: Plenum Press, New York (1990). D.SHIBATA, M.TOKUNAGA, N.SAGAKI, K.NAMBA, Am.J.of Clin. path. 95 536-539 (1991). N.K. SAKSENA, D.E.DWYER, F.B. SINOUSSI, J . i n f . d i s e a s e s . 1 6 4 1021-1022 (1991). D.E.DWYER, N.K.SAKSENA, Mol. cell. probes. In press (1992). F.BARRE-SINOUSSI, J.C.CHERMANN, F.REY, M.T.NUGEYRE, S.CHAMARET, J.GRUEST, C.DAUGUET, C.AXLER BLIN, F.BRUNVEZINET, C.ROUZIOUX, W.ROZENBAUM, L.MONTAGNIER, S c i e n c e . 2 0 0 868-872 (1983). F.CLAVEL, D.GUETARD, F.BRUN-VEZINET, S.CHAMARET, M.A.REY, M.D.SANTOS-FERREIRA, A.GLAURENT, J.C.CHAMPALIMAUD, L.MONTAGNIER, Science.233 343 (1986). R.F.DOOLITTLE, D.F.FENZ, M.S.JOHNSON, M . A . M c C L U R E , Q . R e v . Biol. 6__441 (1989). R.F.DOOLITTLE, Nature (London). 3 3 9 3 3 8 (1989). V.m. HIRCH, R.A.OLMSTEAD, M.MURPHEY-CORB, R.H.PURCELL, P.R. JOHNSON, Nature (london). 3 3 9 3 3 9 (1989). T.F.SMITH, A.SRINIVASAN, G.SCHOCHETMAN, M.MARCUS, G.MYERS, Nature ( l o n d o n ) . 3 3 9 5 7 3 (1988). S.YOKOYAMA.L.CHUNG,and T.GOJOBORI,MoI.BioI.Envol.5237(1988). P.N.FULTZ, H.M.McCLURE, D.C.ANDERSON, R.B.SWENSON, R.ANAD, A. SRINIVASAN, Proc. Natl.Acad. Sci. USA. 8_/35286 (1986). K.I.TSUJIMOTO, R.W.COOPER, S.KODAMA, M. FUKASAWA, T. MIURA, Y. T.OHTA, T.ISHIKAWA, M. NAKAI, E.FROST, G.E.ROELANTS, J.ROFFI, M.HAYAMI, J . V i r o l . 6 2 4 0 4 4 (1988). T.HUET, R.CHEYNIER, A.MEYERHANS, G.E.ROELANTS, S.WAIN-HOBSON, Nature (London) 3 4 5 3 5 6 (1990). Y.T.OHTA, H.MOSUDA, K.I.TSUJIMOTO, T.ISHIKAWA, S.KODAMA, M. MORIKAWA, M.NAKAI, S.HONJO, Int. J. Cancer.4_! 155 (1988). M . D . D A N I E L , N . L . L E T W I N , N.W.KING, M. KANNAGI, P.K.SEHGAL, R.D. HUNT, P.J.KANKI, M.ESSEX, R.C.DESROSIERS, Science. 228 1201 (1985) 15. B. HIRT, Mol. Biol. 2 6 3 6 5 (1967). E . A . M A G A and T.RICHARDSON, Biotechniques. 2 1 0 3 - 1 0 4 (1991).
1984
18. 19.
PcR Amplification of Large Genomic Fragments
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S . D E W H U R S T , J . E . E M B R E S T O N , D . C . A N D E R S O N , J . I . M U L L I N S , P.N. FULTZ, N a t u r e (London). 345 6 3 6 - 6 4 0 (1990). J.SAMBROOK, E.F.FRITSH, T.MANIATIS, Cold spring harbor l a b o r a t o r y p r e s s 1-3 (1989).
PCR
AMPLIFICATION OF LARGE GENOMIC FRAGMENTS IMMUNODEFICIENCY VIRUS INFECTED
Nitin Paul
Pergamon Press
FROM CELL
HUMAN LINES
AND
SIMIAN
K. S a k s e n a * @ , D o m i n i c E. Dwyer***, Michaela M~ller**, Durand****, Vincent Herv~****, Marie-Claude Lang** Fran~oise Barr~-Sinoussi.**
Jean and
* S U N Y , H S C . D e p a r t m e n t of M e d i c i n e , R o o m 1 3 0 5 , W S K , S y r a c u s e , N . Y . 1 3 2 0 3 . * * I n s t i t u t e Pasteur, Biologie des Retrovirus, 25 Rue du Dr. Roux, Paris France. * * * I n s t i t u t e for Clinical pathology and Medical Research, W e s t m e a d Hospital, W e s t m e a d N3W 2145, Australia. * * * * I n s t i t u t e Pasteur, Bangui, Central African Republic. (Received in final form April i0, 1992) Snmmary
P o l y m e r a s e chain reaction (PCR) has been used to a m p l i f y the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from W e s t e r n Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from h i g h l y conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have a m p l i f i e d by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from i2.3 kb from gag pol and env genes have been s u c c e s s f u l l y amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a h i g h l y conserved stretch of the viral genome. Since the onset of PCR as a tool for amplifying DNA, various c o n d i t i o n s and parameters have been defined for its o p t i m i z a t i o n (i). Now DNA can be amplified from a fixed biological material (2). C o n s i d e r a b l e progress has been made in m o l e c u l a r biology using PCR as a technique for studying various genes from diverse p r o c a r y o t e s and eucaryotes. Presently, PCR is being used r o u t i n e l y to study human and animal retroviruses p a r t i c u l a r l y their genomic variability. This has become the most dependable t e c h n i q u e because of its high efficiency and sensitivity, provided appropriate p r e c a u t i o n s are taken to avoid false positive reactions (3, 4). Ever since the discovery of the human i m m u n o d e f i c i e n c y virus 1 and 2 (HIV-I and HIV-2) as causative agents of AIDS (5, 6)
@ A u t h o r to whom correspondence should be addressed 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
type
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PCR Amplification of Large Genomic Fragments
Vol. 50, No. 25, 1992
considerable progress has been made in identifying and characterizing AIDS like viruses (SIV) from various nonhuman primates (7-11). Simian immunodeficiency viruses are primate lentiviruses that share a common ancestry with human immundeficiency viruses (HIV-I and HIV-2) (12-16) and thus it has become important to study more simian i m m u n o d e f i c i e n c y viruses for b e t t e r u n d e r s t a n d i n g of the biological, g e n e t i c and also the phylogentic relationships they share with the Human i m m u n o d e f i c i e n c y virus type-i and type-2. The H I V and SIV may have low copy number in infected cells and to clone the entire viral genome becomes a tedious task and exorbitant in terms of cost. Amplifying short length genomic fragments by PCR is also inefficient and not cost effective. C o n s i d e r i n g these facts, amplifying large genomic f r a g m e n t s w o u l d not only p r o v i d e complete genetic information harbored in the viral genomes but will be less time c o n s u m i n g and m o r e cost effective and would m i n i m i z e problems of PCR c o n t a m i n a t i o n by reducing the number of reactions necessary to o b t a i n genomic m a t e r i a l for c l o n i n g and sequencing. Recent reports (17) have described the a m p l i f i c a t i o n of 5-9 kb gene f r a g m e n t s of B - C a e s i n gene cloned in a plasmid. C r e a t i o n of an i n f e c t i o u s clone of SIVsmm by PCR has also been d e s c r i b e d (18). There are no reports on the optimization of amplification conditions necessary for successfully obtaining large genomic f r a g m e n t s from a retrovirus (HIV and SIV) infected stock. In the p r e s e n t investigation we have defined and o p t i m i z e d the c o n d i t i o n s for the a m p l i f i c a t i o n of large g e n o m i c f r a g m e n t s (i5.5 kb) d i r e c t l y from the HIV2 and SIV infected Molt4 clone-8 cell line, and not from the genes cloned into plasmids. We describe the p o t e n t i a l of PCR in extracting more genetic i n f o r m a t i o n from these p a t h o g e i c viruses in a shorter time and cost e f f e c t i v e manner.
Materials
and Methods
Animals: The A f r i c a n monkeys (Erythrocebus) used in this study were w i l d caught in W e s t e r n Africa in Senegal at the I n s t i t u t e Pasteur. All the animals were healthy and asymptomatic. P e r i p h e r a l blood I y m p h o c y t e s (PBL) from monkeys were sampled and c u l t u r e d w i t h Molt-4 clone 8 cells. Two asymptomatic, seronegative adult females (89005 and 89008 sampled for PBL on 4.26.1989) w e r e used for t h e s e observations. Cell lines and virus culture: PBLs were isolated on p e r c o l l and co-cultivated with C8166 cells in the presence of phytohemagglutinin (PHA). Subsequently, Molt-4 clone 8 cells were used as target cells for sir infection. Culture was done in RPMI 1640 medium supplemented with i00 IU/ml penicillin, 100ug/ml streptomycin and 10% fetal calf serum (FCS). Cultures were maintained at 37°C in a 5% CO 2 incubator and the m e d i u m was r e n e w e d twice a week. The appearance of syncytia s u g g e s t e d the p r e s e n c e of SIV and cytopathic effects (CPE) were o b s e r v e d under light microscopy. Also for HIV-2 (ROD) we used Molt-4 clone 8 cells w h i c h were cultured and h a r v e s t e d after two p a s s a g e s when c y t o p a t h i c effects were visible under light microscopy. U n i n f e c t e d
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PCR Amplification of Large Genomic Fragments
Molt-4 clone 8 cells were maintained parallel as a negative control.
in
continuous
1975
culture
in
Reverse Transcriptase (RT) activity: We measured the DNA p o l y m e r a s e activity [poly (A) dependent] in 50ul of 50mM tris hydrochloride (pH7.5), 5mM dithiothreitol, 100mM KCI, 10mM M a g n e s i u m chloride, 10mM[3H]dTTP, 0.1% Triton X-100 c o n t a i n i n g 2ul of poly (A), 0.4ug of oligo (dT), and an a p p r o p r i a t e amount of virions. The reaction was carried out at 37°C for 1 hour. The polymerized [3H]dTTP was precipitated with 10% t r i c h l o r o a c e t i c acid, c o l l e c t e d on a membrane filter (Millipore Corp., Bedford, Mass), w a s h e d with 10% trichloroacetic acid and ethanol, air dried, and counted in a liquid scintillation counter (5). Electron Microscopy: Virus infected Molt-4 clone 8 cells were fixed in g l u t a c a c o d y l a t e and osmium tetraoxide, and then embedded in araldite. These sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope. Extraction and analysis of uninteqrated viral DNA: U n i n t e g r a t e d HIV-2 (ROD) and SIV DNA was extracted following the m e t h o d of Hirt (15). Briefly, aliquots of 4x106 cells were lysed at 65°C in 0.6% sodium dodecyl sulfate (SDS). Hirt supernatants were e x t r a c t e d twice with phonel, once chloroform and p r e c i p i t a t e d with ethanol. All the cell cultures and the DNA extractions for HIV-2 and SIV were c a r r i e d out under separate P2 biosafety hoods in order to avoid carry over problems. The DNA samples were a l i q u o t e d under the UV s t e r i l i z e d biosafety hoods. Before p r o c e e d i n g for PCR, some of the DNA extracts were electrophoresed in 0.8% tris borate (pH 8.0) agarose gels and transferred to nylon m e m b r a n e filters (Amersham International, UK). Filters were p r e h y b r i d i z e d at 42°C in a solution containing 5xSSC (lxSSC is 0.015M sodium citrate plus o.15M NaCl), 50% formamide, 1% SDS, 3x Denhardt solution, 100ug of d e n a t u r e d salmon sperm DNA/ml and SuM EDTA (pH 8.0). H y b r i d i z a t i o n was done overnight with 2x106 cpm of [alpha-32p] labelled DNA probe per ml (specificity activity, 2x108 to 5xl0Ucpm/ug), and the filters were washed under a g i t a t i o n in 0.2% SSC, 0.1% SDS at 60-65°C. Washed filters were blotted dry, w r a p p e d in saran wrap and subjected to a u t o r a d i o g r a p h y with X-omat i n t e n s i f y i n g screens (Eastman Kodak Co., Rochester, N.Y.) (19). Standard polymerase chain reaction: PCR were p e r f o r m e d in 500ul Eppendorf tubes in a total volume of i00 ul c o n t a i n i n g the following: 10mM Tris HCI (pH 8.3), 50 mM KCI, 1.5 mM MgC12, 0.01% w/v gelatin, 200 mM each of dATP, dCTP, dGTP, dTTP, 2mM of each primer, lug of DNA template, 3U of taq p o l y m e r a s e (Cetus corp, Norwalk, CT). Reactions were overlaid with 60ul of mineral oil and a m p l i f i e d for 35 cycles using an automated t e m p e r a t u r e cycler (LEP Thermocycler, UK). We changed the extension time to f a c i l i t a t e the a m p l i f i c a t i o n of fragments of different sizes. Specific primer sequences and their genomic locations are listed in Table II. i) Amplifyinq ikb-2kb fraqments: 95°C for 5 min., 55°c for 1 min. and 72°C for 2 min. 95°C for 30 sec., 55°C for 1 min. and 72°C for 2 min. 95°C for 30 sec., 55°C for 1 min. and 72°C for 7 min.
(i cycle) (33 cycles) (i cycle)
1976
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PCR Amplification of Large Genomic Fragments
2) Amplifyinq fraqments > 3kb: 9 5 ° C for 5 min., 5 5 ° c for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min. 9 5 ° C f o r 30 sec., 55°C for 1 min.
and 72°C and 7 2 ° C and 7 2 ° C
for for for
4 min. 4 min. i0 min.
3) Amplifyinq fraqments > 4kb: 9 5 ° C for 5 min., 5 5 ° c for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min. 9 5 ° C f o r 30 sec., 5 5 ° C for 1 min.
and 72°C and 72°C and 72°C
for for for
6 min. (cycle) 6 m i n (33 cycles) i0 m i n (33 cycles)
(i cycle) (35 cycles) (i cycle)
A n a l y s i s of PCR products: T w e n t y m i c r o l i t e r s of P C R a m p l i f i e d p r o d u c t w e r e a n a l y z e d on 0.8% a g a r o s e gel. A f t e r e l e c t r o p h o r e s i s for 1 h o u r at 150V, the D N A w a s v i e w e d u n d e r U V t r a n s i l l u m i n a t o r . D N A w a s t r a n s f e r r e d on t h e N y l o n m e m b r a n e a n d h y b r i d i z e d a c c o r d i n g t o t h e p r o t o c o l as d e s c i b e d above. Sequence
SI.No. 1 2 3 4 7 8 9 i0 ii 12 13 14 15 16 17 18 19 20 The
Primer
Table I of o l i g o n u c l e o t i d e P r i m e r s
Location
Sequence
5'-
Used
- - 3'
P2a P2b P7a P7b
env env env env
GCCTGAATAATTGGTATCATTACA TGTAATGATACCAATTATTCAGGC CATTTCCTGATCCGCCAGCTGAT ATCAGCTGGCGGATCAGGAAATG
JFPI JFP2 JFP3a JFP3b JFP4 JFGI
pol pol pol plo pol gag
CAAGCTTAGGAATACCACACCC CAAGCTTGATATACAGAAGTTAGT
JFG2 gag JFEI env JFE2 env D D 916 gag-pol D D 939 gag-pol D D 6512 gag-pol DDI0169 env-ltr DDI0269 env-ltr sequence before the
in P C R
AAAGCTT/GGGTACCAGCACACAAAGG AAAGCTT/TGTGTGCTGGTACCCA
AAAGCTT/GCTCCTTCCCTTTCCA
Consensus between SIVmaC/ HIV-2
HIV-2 SIVsm SIVmac SIVagm TYO-I
AGAATTCCGCCCGAACAGGGACTTGA TGAATTCAGGCCGTCAGCATTTCTTC CGAATTC/AAAAAGTGTTGCTACCA
CGAATTC/CAATAATCTTTGTCACA GGCAGTAAGGGCGGCAGGAAC AACCACGACGGAGTGCTCCTA GGAAATCCTCTCTCAGCTATACCG TGAATTGTGCCCAGCACCGGCCAAGTG ATCTCTCCTAGTCGCCGCCTGGTGAC s l a s h (/) i n d i c a t e s t h e l i n k e r
HIV-2/ SIVmac HIV-2 HIV-2 HIV-2
ROD NIH-2 ISY
sequence
Results E l e c t r o n microscopy: T h e p r e s e n c e of v i r u s in t h e t w o i n f e c t e d m o n k e y s , 8 9 0 0 5 a n d 8 9 0 0 8 (fig. IA a n d IB) w a s c o n f i r m e d i n i t i a l l y by e l e c t r o n m i c r o s c o p y . M o l t - 4 a n d c l o n e 8 c e l l s r e v e a l e d H I V / S I V like retroviral particles. Viral particles, after 6 days of culture, c o u l d be s e e n b u d d i n g f r o m t h e c e l l s u r f a c e in c a s e of 8 9 0 0 5 i n f e c t e d c e l l s (fig. IA) and s e v e r e C P E w i t h m a s s i v e c e l l death and high viral load w a s s e e n in c a s e of 8 9 0 0 8 infected cells, a l s o a f t e r 6 d a y s of c u l t u r e (fig. IB). T h i s i n d i c a t e d t h a t the E r y t h r o c e b u s m o n k e y s h a r b o r e d SIV in t h e i r w i d h a b i t a t . U n d e r light microscopy large syncytia and cell death with large c e l l u l a r s p a c e s w e r e a l s o o b s e r v e d ( r e s u l t s n o t shown).
Vol. 50, No. 25, 1992
PCR Amplification
Viral Consensus Sequence Between Code HIV-2 SIVagm SIVmac SIVsmm
T a b l e II of D N A f r o m I s o l a t e s 89005, SIVmac
Genome
Region
JFG JFPl-3b JFPI-4 JFP2-3b JFP2-4 JFP3a-4
HIV-2 SIVmac HIV-2 (ROD)
PCR Amplification of Large Genomic Fragments
JF E P2b-P7b
DD 916-6442
HIV-2
Amplification
Position
Fragment Length(bp)
gag pol pol pol pol pol
315-1600 2610-3960 2610-4765 3100-3960 3100-4765 3935-4765
1285 1350 2155 860 1665 830
env env
6230-8430 6825-8430
800 1600
gag-pol
916-6442
HIV-2 NIH-2 HIV-2 ISY
89008,
5200
1977
(ROD)
and
Products
89005
89008
HIV-2
SImac
-* + + + + +
-NS +NS +NS +NS +NS +NS
-* + + + + +
-* + + +
+
+
+
NT
+
+
+
NT
5500 DD65212-I0171
env-LTR
6512-10171
3500 3700
H y b r i d i z a t i o n w a s d o n e w i t h H I V - 2 (ROD) e n t i r e probe (gag-pol-env-ltr) under high stringency conditions and with stringent washes
NS
- Not
Shown
NT - N o t T e s t e d
* T e s t e d n e g a t i v e b e c a u s e H I V 2 (ROD) p r o b e w a s l a c k i n g g a g r e g i o n (315-1600) + - Amplification and Hybridization positive
Reverse
Transcriptase activity: A f t e r 6 d a y s of c u l t u r e of 8 9 0 0 5 and 89008 infected Molt-4 clone 8 cells showed low t i t e r s of r e v e r s e R T a c t i v i t y in t h e c u l t u r e s u p e r n a t a n t s . In c o n t r a s t , t h e S I V m a c a n d H I V - 2 (ROD) i n f e c t e d M o l t - 4 c l o n e 8 c e l l s s h o w e d h i g h t i t e r s of R T a c t i v i t y at the end of s e c o n d p a s s a g e ( r e s u l t s n o t shown)
Southern blot hybridization: Before PCR amplification, the Hirt DNA preparation from infected cells was hybridized with SIVmac (R9) p r o b e c o n t a i n i n g t h e e n t i r e v i r a l g e n o m e . T h e D N A f r o m M o l t - 4 c l o n e 8 c e l l s i n f e c t e d w i t h S I V i s o l a t e s (89005 a n d 89008) s h o w e d hybridization s i g n a l s , t h e r e b y i n d i c a t i n g t h e p r e s e n c e of v i r a l DNA. T h e s e o b s e r v a t i o n s c o n c u r r e d w i t h the r e s u l t s o b t a i n e d w i t h electron microscopy. The DNA from the isolate 89008 also showed t h e p r e s e n c e of u n i n t e g r a t e d v i r a l D N A of a b o u t 9 k b (fig. 2). Similarly, DNA from Molt-4 clone 8 infected with SIVmac and HIV-2 (ROD) also showed good hybridization signals with SIVmac (R9) probe.
1978
PCR Amplification of Large Genomic Fragments
FIG.1A
and
Vol. 50, No. 25, 1992
1B
Electron microspy of 8 9 0 0 5 and 89008 infected Molt-4 clone 8 cells. A. SIV (89005) infected Molt-4 clone 8 cells showing b u d d i n g of v i r u s p a r t i c l e f r o m t h e c e l l s u r f a c e a f t e r 6 days of i n f e c t i o n . M a g x l 6 0 0 . 0 0 0 . B. S I V (89008) i n f e c t e d Molt-4 clone 8 cells showing mature virus particles after 6 days of i n f e c t i o n . M a g x 200,000. v i r u s p a r t i c l e s c a n be s e e n i n s i d e t h e d e a d cells. (Arrows indicate the budding and the mature virus particles.)
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
1979
05
1 A I
C D
E
DNA
FIG.
2
Southern blot hybridization H i r t p r e p a r a t i o n of viral DNA f r o m M o l t - 4 c l o n e 8 c e l l s infected with 89005, 89008, SIVmac and HIV-2 (ROD). S I V m a c (R9), c o n t a i n i n g the e n t i r e v i r a l genome, h a s b e e n u s e d as a probe. L a n e A. D N A f r o m 89005 i n f e c t e d cells. L a n e B. D N A f r o m 89008 i n f e c t e d cells. L a n e s C a n d D. D N A f r o m S I V m a c and HIV-2 (ROD) i n f e c t e d cells.
PCR amplification: We h a v e s i m p l i f i e d the P C R a m p l i f i c a t i o n of long genomic fragments from HIV-2 (ROD) a n d S I V i n f e c t e d cell lines by using consensus primer sequences taken from highly c o n s e r v e d r e g i o n s b e t w e e n S I V m a c and HIV-2. F i g u r e 3 s h o w s t h a t we have amplified D N A f r o m the 89005 isolate. The result of P C R a m p l i f i c a t i o n w i t h D N A f r o m 89008 h a v e b e e n t a b u l a t e d in t a b l e II. In o r d e r to b e t t e r o p t i m i z e the c o n d i t i o n s for a m p l i f i c a t i o n of l a r g e g e n o m i c s e g m e n t s of r e t r o v i r a l g e n e s we h a v e a s s a y e d v a r i o u s overlapping oligonucleotides extending over the entire viral g e n o m e (LTR, gag, pol,and env) (Table i).
1980
PCR Amplification of Large Genomic Fragments
A
B
C
O
E
F
O
P9.11
,,
O
H
~
J
K
Vol. 50, No. 25, 1992
L
M
N
O
el
~
.
+
.
X +1.
2
3
~e FIG • 3 P C R a m p l i f i c a t i o n of g e n o m i c f r a g m e n t s r a n g i n g f r o m 8 0 0 2 1 5 0 b p f r o m E r y t h r o c e b u s i s o l a t e (89005), HIV-2 (ROD) a n d S I V m a c by u s i n g c o n s e n s u s p r i m e r s e q u e n c e s b e t w e e n SIVmac, H I V - 2 a n d SIVsm. L A N E S A , B , C , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F G (1300bp) L A N E S D , E , F , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P l - 3 B ( 1 3 5 0 b p ) L A N E S G , H , I , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 2 - 3 B (860bp) L A N E S J , K , L , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P I - 4 (2150bp) L A N E S M , N , O , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 2 - 4 (1660bp) L A N E S P , Q , R , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F P 3 - 4 (8200bp) L A N E S S , T , U , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r J F E (800bp) L A N E S V , X , Y , - H I V - 2 S I V m a c and 8 9 0 5 - P r i m e r P 2 - P 7 (1600bp) L A N E S 1 , 2 , 3 , - N e g a t i v e c o n t r o l s (No DNA: D N A f r o m n o n i n f e c t e d M o l t 4 c l o n e 8 cells, and no p r i m e r control. H y b r i d i z a t i o n was c a r r i e d out w i t h HIV-2 (ROD) probe, l a c k i n g the g a g region, u n d e r h i g h s t r i n g e n c y c o n d i t i o n s
(65°c).
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
1981
Using this strategy we have successfully a m p l i f i e d viral gene fragments ranging from 830bp-2155bp (fig.3 and Table II). The e x t e n s i o n cycle in this case was 1-3 minutes. To be more sure of amplification efficiency of fragments between 1000-2000bp we d e s i g n e d several primer pairs in gag and pol regions along with some o v e r l a p p i n g primer pairs which amplified 1285bp in gag and 1350, 860, 2155, 1665 and 830bp in pol gene. All of them were hybridization positive with HIV-2 (ROD) except the SIVmac. C o n s i d e r i n g the fact that HIV-2ROD and SIVmac are 35% d i v e r g e n t from each other, the absence of signals under high s t r i n g e n c y hybridization conditions can be expected. These results d e m o n s t r a t e that the fragments ranging from 8 0 0 - 2 1 5 5 b p may be e f f i c i e n t l y amplified by using extension time between 1-3 minutes per cycle. No h y b r i d i z a t i o n specific a m p l i f i c a t i o n was seen with primer JFE and P2-P7 (fig.3 Lanes S,T,U and V,W,X). The a m p l i f i e d p r o d u c t s were nonspecific amplicons. Similarly, gag fragments (1285bp, Lanes A,B and C; fig. 3) did not show any hybridization. This is because of the fact that the HIV-2 (ROD) probe lacked the gag region. In another set of experiments we amplified the entire viral genome of a p p r o x i m a t e l y <9kb in two halves of 3.5 and 5.2kb. One half spans from within the U3 region of 5'LTR into pol gene and an o v e r l a p p i n g fragment spans 3700bp from the pol gene into U5 region of 3'LTR (fig. 4; Table II). In each case (fig.3 and fig.4) the a m p l i f i e d fragments when hybridized with specific genomic probes revealed the specificity of PCR.
DISCUSSION
We have d e s c r i b e d a convenient way of generating long genomic DNA fragments, from cells infected with HIV-2 (ROD) and SIV, by PCR. Our data emphasizes the potential of PCR in h a r v e s t i n g more g e n e t i c information from these complex group of viruses, which have very low copy number of viral DNA, in a shorter time. The c o n d i t i o n s defined here are cost effective and highly efficient. Several approaches have been adopted for g e n e r a t i n g short genomic fragments from viral genes, but all this is very time consuming. H a v i n g a little starting substrate material for a m p l i f i c a t i o n is also a c o n s t r a i n t in the study of these retroviruses. This makes cloning of retroviruses in conventional phages more tedious and time consuming. Long range PCR can facilitate the cloning and s e q u e n c i n g of retroviral genes in a much simpler way and this we have demonstrated by amplifying the entire genome of SIV (Erythrocebus) and HIV-2 (ROD) in two halves. A similar approach has been taken with another SIV, SIVSmmPBjI4 (18). In our analyses, it is clear that the extension time is important for e f f i c i e n t amplification of large genomic fragments. We allow one m i n u t e per kilobase as more prolonged e x t e n s i o n times favor the f o r m a t i o n of nonspecific bands. The q u a n t i t y of a m p l i f i e d large fragments obtained from samples with low copy numbers of DNA may be low, but it is usually sufficient for cloning and sequencing. If the yield is low, the PCR fragments can be r e a m p l i f i e d with the same primers or with nested primers, although case must be taken to avoid contamination (3). Occasionally, p r o b l e m s are encountered with nonspecific bands or primers dimers
1982
PCR Amplification of Large Genomic Fragments
A
B
FIG.
Vol. 50, No. 25, 1992
C
4
PCR a m p l i f i c a t i o n of env fragment (3.5 kb) and g a g - p o l fragment (5.2 kb) from DNA of Erythrocebus isolate 89005 by u s i n g d i s t a n t l y located p r i m e r s conserved in HIV-2. L a n e M - M o l e c u l a r w e i g h t m a r k e r (Lambda H i n d III d i g e s t e d ) A- D N A f r o m 89005 i n f e c t e d c e l l s (Lane A - 8 9 0 0 5 D N A a n d L a n e D - H I V - 2 DNA) s h o w i n g t h e a m p l i f i c a t i o n of 3.7 kb e n v f r a g m e n t . L a n e s B and C are n e g a t i v e c o n t r o l s . B e l o w are the hybridization results. B- D N A from 89005 and H I V - 2 i n f e c t e d cells showing amplification of 5.2 k b g a g - p o l fragment (Lane- A 89005 and lane -D HIV-2 DNA). Lanes B and C are negative controls. B e l o w are the h y b r i d i z a t i o n results. C- A l s o s h o w s t h e amplification of 89005 DNA f r o m i n f e c t e d cells. H e r e t h e 3.5 kb e n v f r a g m e n t w a s a m p l i f i e d by u s i n g a n e s t e d p r i m e r (see T a b l e I a n d II). On t h e r i g h t h a n d side are the h y b r i d i z a t i o n r e s u l t s . In all the cases the HIV-2 (ROD) probe has been used for hybridization under stringent conditions.
when reamplifying PCR products to o b t a i n enough material for s e q u e n c i n g . T h i s is p r e s u m a b l y d u e to the p r e s e n c e (often n o t s e e n on t h e gels) of non specific amplified material, a n d c a n be m i n i m i z e d by p u r i f y i n g the b a n d s of i n t e r e s t f r o m gels, e n s u r i n g t h a t t h e a l i q u o t s f r o m the p r e v i o u s P C R are h i g h l y d i l u t e d , a n d by e n s u r i n g t h e n e s t e d p r i m e r s are h i g h l y s p e c i f i c . W i t h the l o n g e r e x t e n s i o n t i m e s r e q u i r e d for large f r a g m e n t s it is i m p o r t a n t not to u s e t o o m a n y c y c l e s (eg. less t h a n 25) so t h a t n o n s p e c i f i c b a n d s a n d loss of taq p o l y m e r a s e a c t i v i t y are limited. T h i s is especially important when performing nested PCR. Excessive taq polymerse (eg. m o r e than 2-4 U n i t s per reaction) is a l s o an a v o i d a b l e c a u s e of n o n s p e c i f i c bands, e s p e c i a l l y w h e n a m p l i f y i n g f r o m m a t e r i a l c o n t a i n i n g low levels of virus. T h e o p t i m a l a m o u n t of e n z y m e n e e d s to be a s c e r t a i n e d w i t h e a c h p r i m e r pair, b u t in g e n e r a l 1 u n i t or less is all t h a t is n e c e s s a r y . Primer selection is o b v i o u s l y crucial to the success of PCR. A l t h o u g h t h e g e n e t i c v a r i a t i o n and h i g h m u t a t i o n r a t e s of H I V a n d S I V a r e w e l l k n o w n (9,12,14,18) the s e l e c t i o n of p r i m e r s u s e d in
Vol. 50, No. 25, 1992
PCR Amplification of Large Genomic Fragments
1983
these e x p e r i m e n t s in highly conserved regions allowed r e a s o n a b l y high stringency conditions to be used. In our experience, o c c a s i o n a l m i s m a t c h e s in oligonucleotide primers did not affect the a m p l i f i c a t i o n of shorter fragments (between 100-700bp), but could affect the amplification of larger fragments (>800bp). Our analyses are useful as they demonstrate the value of PCR in a m p l i f y i n g large viral genomic fragments, from low copy genomic material, for cloning and sequencing. This may prove useful not only to retrovirologist, but can also be applied to study other viruses and lower organisms. This will facilitate the u n d e r s t a n d i n g of molecular and biological c o m p l e x i t y of these organisms in a shorter time and in a cost effective m a n n e r and will also pave the way for defining new lentiviruses. Acknowledgements
NKS is thankful to CIES, Paris (France) for financial assistance. We are indebted to Drs Luc Montagnier and Francois Clavel for HIV-2 (ROD) probe. References
i.
2 3. 4. 5.
6.
7. 8. 9. i0. ii. 12. 13.
14. 15. 16. 17.
B.J.POIES, G.D.EHRLICH, B.C.BYRNE, K.WELLS, S.KWOK, J.SNINSKY,L.M. de la Maza and E.M.Peterson (eds). Medical V i r o l o g y 9: Plenum Press, New York (1990). D.SHIBATA, M.TOKUNAGA, N.SAGAKI, K.NAMBA, Am.J.of Clin. path. 95 536-539 (1991). N.K. SAKSENA, D.E.DWYER, F.B. SINOUSSI, J . i n f . d i s e a s e s . 1 6 4 1021-1022 (1991). D.E.DWYER, N.K.SAKSENA, Mol. cell. probes. In press (1992). F.BARRE-SINOUSSI, J.C.CHERMANN, F.REY, M.T.NUGEYRE, S.CHAMARET, J.GRUEST, C.DAUGUET, C.AXLER BLIN, F.BRUNVEZINET, C.ROUZIOUX, W.ROZENBAUM, L.MONTAGNIER, S c i e n c e . 2 0 0 868-872 (1983). F.CLAVEL, D.GUETARD, F.BRUN-VEZINET, S.CHAMARET, M.A.REY, M.D.SANTOS-FERREIRA, A.GLAURENT, J.C.CHAMPALIMAUD, L.MONTAGNIER, Science.233 343 (1986). R.F.DOOLITTLE, D.F.FENZ, M.S.JOHNSON, M . A . M c C L U R E , Q . R e v . Biol. 6__441 (1989). R.F.DOOLITTLE, Nature (London). 3 3 9 3 3 8 (1989). V.m. HIRCH, R.A.OLMSTEAD, M.MURPHEY-CORB, R.H.PURCELL, P.R. JOHNSON, Nature (london). 3 3 9 3 3 9 (1989). T.F.SMITH, A.SRINIVASAN, G.SCHOCHETMAN, M.MARCUS, G.MYERS, Nature ( l o n d o n ) . 3 3 9 5 7 3 (1988). S.YOKOYAMA.L.CHUNG,and T.GOJOBORI,MoI.BioI.Envol.5237(1988). P.N.FULTZ, H.M.McCLURE, D.C.ANDERSON, R.B.SWENSON, R.ANAD, A. SRINIVASAN, Proc. Natl.Acad. Sci. USA. 8_/35286 (1986). K.I.TSUJIMOTO, R.W.COOPER, S.KODAMA, M. FUKASAWA, T. MIURA, Y. T.OHTA, T.ISHIKAWA, M. NAKAI, E.FROST, G.E.ROELANTS, J.ROFFI, M.HAYAMI, J . V i r o l . 6 2 4 0 4 4 (1988). T.HUET, R.CHEYNIER, A.MEYERHANS, G.E.ROELANTS, S.WAIN-HOBSON, Nature (London) 3 4 5 3 5 6 (1990). Y.T.OHTA, H.MOSUDA, K.I.TSUJIMOTO, T.ISHIKAWA, S.KODAMA, M. MORIKAWA, M.NAKAI, S.HONJO, Int. J. Cancer.4_! 155 (1988). M . D . D A N I E L , N . L . L E T W I N , N.W.KING, M. KANNAGI, P.K.SEHGAL, R.D. HUNT, P.J.KANKI, M.ESSEX, R.C.DESROSIERS, Science. 228 1201 (1985) 15. B. HIRT, Mol. Biol. 2 6 3 6 5 (1967). E . A . M A G A and T.RICHARDSON, Biotechniques. 2 1 0 3 - 1 0 4 (1991).
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18. 19.
PcR Amplification of Large Genomic Fragments
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S . D E W H U R S T , J . E . E M B R E S T O N , D . C . A N D E R S O N , J . I . M U L L I N S , P.N. FULTZ, N a t u r e (London). 345 6 3 6 - 6 4 0 (1990). J.SAMBROOK, E.F.FRITSH, T.MANIATIS, Cold spring harbor l a b o r a t o r y p r e s s 1-3 (1989).