33

Journalof VirologicalMethods, 28 (1990) 33-46

Ekvier VIRMET 00997

The use of primers from highly conserved pal regions to identify uncharacterized retroviruses by the polymerase chain reaction Lawrence A. Donehowerl,

Robert C. Bohannonl, Richard A. Gibbs2

Richard J. Ford3, and

‘Division of Molecular Virology and 21n.&ute for Molecular Genetics, Baylor College of Medicine, and ‘Department

of Molecular Pathology, M.D. Anderson

Cancer Center, Houston,

Texas, 77030

(Accepted 30 November 1989)

Two degenerate oligonucleotide primers derived from regions of pal conserved among retroviruses have been synthesized. Polymerase chain reactions utilizing these primers amplify a 135-bp pol fragment in every retrovirus DNA tested to date. Ihe polymerase chain reaction has been linked to a reverse tramcriptase step so that a pal-specific DNA fragment can be obtained from a moderate amount of a purified retrovirus or viral RNA. The identity of an unknown retrovirus can be determined by sequencing of the amplified fragment following molecular cloning. This procedure was tested on an unidentified (non-HIV) retrovirus expressed by a B-cell lymphoma line obtained from an AIDS patient. Our PCR assay identified the retrovirus as being highly similar to MasonPfizer monkey virus (MPMV) and simian retrovirus 1, which are closely related immunosuppressive type D viruses that cause simian AIDS. Retrovirus; Polymerase chain reaction; identification; Simian type D retrovirus

Reverse transcriptase

assay; Retrovirus

Intruduction

The polymerase chain reaction (PCR) has emerged as a powerful and sensitive procedure for the amplification of specific nucleic acid sequences (Saiki et al., 1985; Correspondence to: Lawrence A. Donehower, Division of Molecular Virology, Baylor College of Medicine, One. Baylor Plaza, Houston, TX 77030.

0166-0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

34

Mullis and Faloona, 1987). PCR methods have been particularly useful in the detection of human retroviruses in the blood and tissues of infected individuals (Ou et al., 1988; Murakawa et al., 1988; Bell and Ratner, 1989; Kwok and Sninsky, 1989). It has been hypothesized that a number of disease-associated human retroviruses remain undiscovered. PCR technologies may provide a highly sensitive means of detecting and identifying these agents. Unfortunately, the different members of the retrovirus family are so divergent in sequence that obtaining PCR primers of sufficient length and specificity that will anneal to all retroviral templates is a difficult task. However, two groups have attacked this problem by identifying short segments of sequence in the pol region of all retroviruses and hepadnaviruses which are highly conserved at the amino acid level (Mack and Sninsky, 1988; Shih et al., 1989). They found that relatively short, degenerate primers were capable of amplifying the expected pol fragment. Therefore, the use of the appropriate ‘universal’ primers may enable an investigator to amplify and identify segments of novel retroviruses. One drawback to such an approach is the presence of thousands of endogenous retroviruses and retrotransposons in the genomes of humans (and other eucaryotic species). Amplification of the desired novel retroviral fragments from cellular DNA (or RNA) would be impossible because endogenous sequences anneal to the primer and compete for amplification. In those cases where mature virus can be obtained and purified away from cellular nucleic acids, the amplification of the desired fragment may be possible. In this paper, we describe a technique for obtaining a polspecific DNA fragment from moderate quantities of a purified retrovirus or viral RNA. The purified retrovirus is incubated in an endogenous reverse transcriptase (RT) reaction and an aliquot of this is subjected to PCR utilizing the ‘universal’ retrovirus primers. Alternatively, cDNA is synthesized from purified viral RNA prior to PCR. The resulting 135bp pol fragment can then be molecularly cloned into an appropriate vector prior to sequencing. The universal primer-based PCR procedure was tested on an unknown retroviral isolate obtained from a B-cell lymphoma in an AIDS patient. This method successfully identified the virus as very similar to Mason-Pfizer monkey virus (MPMV) and simian retrovirus 1 (SRV-1). The virus identity was confirmed by immunologic and protein sequencing methods.

Materials and Methods Virus and viral RNA purification

The Prague C strain of Rous sarcoma virus was grown under standard conditions in chicken embryo fibroblasts. Virus-containing medium (120 ml) was clarified by centrifugation at low speed and mixed with 30 ml of SXTNE-sucrose (50% sucrose in 0.5 M NaCl, 100 mM Tris-HCl, pH 7.4, 5 mM EDTA). Five milliliters of 60% sucrose in TNE were placed into six Beckman SW27 centrifuge tubes and were overlaid. with 5 ml of 15% sucrose in TNE and then with 25 ml of virus-con-

35

mining medium in TNE. These step gradients were centrifuged in a Beckman SW27 rotor at 20000 rpm for 2 h at 4°C. Virus bands were collected by syringe at the step gradient boundary, diluted with TNE, and layered onto 25-55% sucrose gradients in SW27 centrifuge tubes. The gradients were centrifuged in an SW27 rotor at 22000 rpm at 4°C for 18 h. Virus bands were removed by syringe and stored in aliquots at -70°C for further experiments. The concentration of virion particles was determined by electron microscopic particle count (Smith and Melnick, 1962). The uncharacterized retrovirus (later shown to be related to MPMV) was harvested from transformed B-cells established in culture from an unusual syncytial form of B-cell lymphoma found in an AIDS patient. One hundred milliliters of virus-containing medium were centrifuged at low speed and filtered through 0.2urn Nalgene filters. Thirty ml of media were layered onto a 5 ml 30% glycerol cushion in Beckman SW28 ultracentrifuge tubes and spun at 27080 rpm for 2 h at 17°C in a Beckman L8-M ultracentrifuge. The pelleted virus was resuspended in 1 ml and layered onto a 25-50% sucrose gradient prior to centrifugation in a Beckman SW41 rotor at 36000 t-pm for 12-14 h at 17°C. Virus bands were visualized by light from a high-intensity lamp, and the band was removed by syringe needle puncture. This virus was pelleted by high speed ultracentrifugation, resuspended in a small volume of water, phenol-chloroform extracted three times, made 0.75 M in sodium acetate, pH 6, and ethanol precipitated, washed, and the RNA pellet dried in a Speed Vat. The RNA was resuspended in a small volume of water. cDNA was synthesized from the viral RNA utilizing the Amersham cDNA synthesis kit. Endogenous

reverse transcriptase reaction

For the endogenous reverse transcriptase reactions, the following RT cocktail was prepared: 50 mM Tris-HCl, pH 8.0,60 mM NaCl, 8 mM MgClz, 0.05% NP40, 2 mM dithiothreitol, 5 OD,, units/ml pd(N), random hexamers (Pharmacia, Cat. No. 27-2166-Ol), and 100 PM each of dATP, dCTP, dGTP, and TTP. Aliquots of this cocktail were stored at -20°C. For each RT reaction, 50 ~1 of cocktail was mixed with 10 t~,lof virus in sucrose-TNE. The reaction mix was incubated at 37°C for 2 h. After completion of incubation, an aliquot (3 ul) was taken for the PCR reaction and the remainder was frozen at -20°C. Polymerase chain reaction with ‘universal’ primers

For PCR from plasmid templates, 1 ng of plasmid was used in each 100 ~1 reaction. For PCR from reverse-transcribed cDNAs in virions, 3 ul of the original 60 ~1 reverse transcription reaction were used. For PCR from cDNAs synthesized from purified viral RNA, 1 u.1of the cDNA was used. PCR conditions and components were as prescribed by the Cetus/Perkin Elmer kit, and reactions were performed in the Perkin Elmer/Cetus DNA Thermal Cycler. For amplification from both plasmid and reverse-transcribed templates, the following program was followed: cycles l-10 - 94”C, 1 min; 37°C 2 min; 72”C, 3 min; cycles 114 - 94°C 30 s; 55”C, 1 min; 72”C, 1 min.

36

Analysis of amplified DNA

One tenth (10 ~1) of each PCR mix was subjected to agarose gel electrophoresis on a 2% agarose gel in the presence of ethidium bromide (0.5 pg/ml) and Trisacetate EDTA buffer (Maniatis et al., 1982). Molecular cloning and sequencing

of amplified fragments

In preparation for molecular cloning of PCR-amplified retroviral fragments, several 100 l.r,lPCRs were performed. These amplified DNAs were subjected to preparative polyacrylamide gel electrophoresis on an 8% gel (1.5 mm thick). The gel was ethidium bromide stained, and the 135-bp retroviral specific band was excised. The DNA fragment was eluted by crushing of the gel fragment (by pushing it through a 16-gauge syringe needle) and shaking overnight in 5 ml of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The solution was centrifuged at low speed (1000 rpm), and the DNA-containing supernatant was passed through a small DEAE cellulose column (DE-52, Sigma) equilibrated in TE. The DNA was eluted from the column in 1 ml 1 M NaCl and ethanol precipitated with 2 ml of ethanol. The precipitate was pelleted by centrifugation, and the DNA pellet rinsed with ethanol, air dried, and resuspended in 10 l.~lTE. For molecular cloning, 3 ~1 of, DNA fragment were ligated (with 200 units T4 DNA ligase; New England Biolabs) in 5 ~1 total volume with 200 ng of BlueScript plasmid vector previously cleaved with SmaI. Ligation was overnight at 10°C. The ligase mixture was then heat inactivated (70°C 15 min) and cleaved with 25 units of SmaI for several hours. This effectively reduces the background of plasmids with no insert fragments. One-fourth of the cleaved ligation mixture was then used to transform 100 ~1 of transformation-competent C600 bacteria according to the Hanahan (1983) procedure. Individual ampicillin-resistant colonies were amplified in L broth-ampicillin, and plasmids were prepared from 1.5 ml bacteria by the method of Birnboim and Doly (1979). Recombinant plasmids were monitored for appropriate size inserts by cleavage with XhoI and XbaI, which cut on either side of the insert in the BlueScript plasmid polylinker region. Plasmids with inserts of the correct size were then directly sequenced using the U.S. Biochemical Corporation Sequenase sequencing kit and procedures for dideoxy sequencing of double-stranded DNA with 35S-labelled oligonucleotides (Sanger et al., 1977; Tabor and Richardson, 1987). Primers complementary to the T3 and T7 promoters located adjacent to the polylinker region in the BlueScript plasmid were used to initiate synthesis of DNA strands for sequencing. Sequencing reactions were subjected to urea polyacrylamide gel electrophoresis and autoradiography. Each clone was sequenced on both strands to reduce the possibility of sequence errors. All computer sequence analysis work was performed on the Baylor College of Medicine Molecular Biology Information Resource. The streamlined user interface EuGene, developed by Thomas Shalom, was used to search GenBank efficiently and to make sequence comparisons. The GenBank search programs were

31

developed by Charles Thomas and Dan Goldman (Lawrence et al., 1986; Lawrence and Goldman, 1988).

Results Design and testing of ‘universal’ retrovirus primers Amino acid sequence comparison has revealed that all retroviruses and retrotransposons share certain core homology regions in their reverse transcriptase coding domains (Toh et al., 1985; Johnson et al., 1986). Two of the most highly conserved regions within pd are only about 100 nucleotides apart and were utilized to derive ‘universal’ PCR primers which might be expected to anneal to all retroviral templates under the appropriate conditions. Fig. 1 she? the amino acid and nucleotide sequences of a number of retroviruses in these two highly conserved regions. In addition, the synthesized degenerate primers obtained from these conserved regions are shown. The 5’ conserved sequence contains the VLPQG motif, which occasionally diverges at the V codon (e.g., MO-MLV and EIAV). The 3’ HTLV-I

GTACIACCCCAAGGG VLPQG

TTTAAA -78 bp- CTTCAG l'ACAl'iXAl'GAC

RTLV-II

GTCCTTCCACAGGGG VLPQG

TTTAAA -78 bp- GTCCAA TACATGGATGAC Y I4 D D FK V Q

HIV-I

GTGCTTCCACAGGGA VLPQG

TGGAAA -78 bp- TATCAA TACATGGATGAT Y K D D WK Y Q

Pr-RSV

GTCTTGCCCCAAGGG VLPQG

ATGACC -78 bp- TTGCAT TATATGGATGAT L H Y M D D KT

MO-KLV

AGACTCCCACAGGGT RLPQG

TTCAAA -78 bp- CTACAG TACGTGGATGAC Y V D D FK L Q

BLV

GTCCTACCTCAAGGC VLPQG

TTCATT -78 bp- GTGTCC TATATGGACGAT v s Y R D D FI

EIAV

TGTTTACCACAAGGA CLPQG

TTCGTG -78 bp- TATCM FV Y Q

MKTV

GTTTTGCCCCAGGGT VLPQG

ATGAAA -70 bp- GTGCAT TACATGGATGAC v H Y U D D MK

FK

5' PRIMER 5'CtcgQatccGTNYTNCCNCA

L

Q

Y

n

D

0

TATATGGATGAT Y M D D

3# PRIMER 3'

3' ATRTACCTRCTRCagCtgetC

5'

Fig. 1. Nucleotide and amino acid comparisons of two highlyconserved segments of representative retroviral pol genes and nucleotidesequence of two synthetic oligonucleotides used for priming poly-

merase chain reactions. Standard single-letter abbreviations are used to designate amino acids, and all nudeotide sequences, except for the 3’ primer, are oriented in the 5’ to 3’ (sense) direction. Primer nucleotides in lowercase letters represent noncomplementary 5’ extensions that contain recognition sequences for BamHI (5’) and SafI (3’) restriction endonucleases. N, Y, and R indicate all four nucleotides, pyrimidines, and purities, respectively, in each position. The retrovirus sequences presented here are as follows: HTLV-I; I-ITLV-II; HIV-l; Pr-RSV, ROW sarcoma virus (Prague C strain); MO-MLV, Moloney murine leukemia virus; BLV, bovine leukemia virus; EIAV, equine infectious anemia virus; and MMTV, mouse mammary tumor virus.

38

123456789M

Fig. 2. Amplification of retroviral DNA target sequences utilizing the ‘universal’ retrovirus primers. One nanogram of plasmid vector each containing a complete retroviral genome was amplified by PCR. Following amplification, an aliquot of each reaction mix was digested with a restriction endonuclease specific for each amplified fragment. Ten microliters of the amplified products were examined by ethidium bromide/agarose gel eiectrophoresis. The arrow indicates the expected 135bp &specific amplified fragment. The following amplified DNAs are shown: (1) pBR322 control; (2) HIV-l; (3) HIVl/S@; (4) HTLV-II; (5) HTLV-IIIDruI; (6) RSV; (7) RSVIRsaI; (8) MO-MLV; (9) MO-MLVIBamHI; (M) 123-bp ladder (Bethesda Research Laboratories).

conserved sequence is the YMDD tetrapeptide, in which the methionine codon has been infrequently substituted with a valine codon (e.g., MO-MLV). The 5’ and 3’ primers contain 14 and 12 nucleotides, respectively, derived from the two conserved sequences. Each primer contains an additional nine nucleotides, six of which comprise a restriction endonuclease cleavage site (BamHI for the 5’ primer and Sal1 for the 3’ primer). These 5’ noncomplementary extensions dramatically increase the efficiency of amplification when attached to short oligonucleotide primers (Mack and Sninsky, 1988; Frohman et al., 1988). Apparently, the extra nucleotides stabilize the priming oligonucleotides. In addition, molecular cloning of the amplified fragment is simplified. To test the ability of the ‘universal’ primers to amplify the targeted retroviral pol segment, we performed PCR on 1 ng of four cloned retroviral DNAs. These retroviruses, HIV-l, HTLV-II, RSV, and MO-MLV, represent widely divergent branches of the retroviral phylogenetic tree (McClure et al., 1988). For the PCR the only departure from standard conditions was to anneal the primers at 37°C during the first ten cycles to optimize priming from relatively short regions of com-

39

plementarity. Following ten PCR cycles, the annealing temperature was increased to 55°C for a further 30 cycles. The amplified DNA fragments were subjected to agarose gel electrophoresis and visualized after ethidium bromide staining. Fig. 2 shows that all four retroviral DNAs produce the expected 135-bp fragment. To demonstrate that the correct sequence was amplified, each fragment was cleaved with a restriction endonuclease specific for that viral sequence. The amplification of the MO-MLV fragment provides an important test for this approach, since both the 5’ primer (2 mismatches) and the 3’ primer (1 mismatch) are not perfectly complementary to the MO-MLV template. The other three retroviral templates should be perfectly matched to at least a subset of both degenerate primers. Polymerase chain reaction with ‘universal’ primers on reverse transcribed virions

To adapt the PCR amplification procedure to purified virions, we utilized a preparation of purified Prague C Rous sarcoma virus as our test substrate. Approximately 7 x lo7 virions in 10 bl phosphate buffered saline were incubated for

1234M

Fig. 3. RT-linked PCR amplification of target sequences directly from purified &ions. Purifled Rous sarcoma virus (7 x 10’ particles) was reverse transcribed in an endogenous reaction. Following reverse transcription, 3 pl(3.5 x 106 particles) were subjected to PCR with the ‘universal retrovints primers. Ten microliters of the amplified products were directly electrophoresed or cleaved with F&I or R.raI (both enzymes should cleave within the RSV amplified fragment) prior to agarose gel electrophoresis. The arrow indicates the expected 135-bp pal-specific fragment. Lane 1 shows a RT-PCR control performed minus virus. Lane 2 shows an RT-PCR performed with RSV. Lanes 3 and 4 contain amplified RSV fragments cleaved with FokI and RsuI, respectively. Lane 5 contains the 123-bp ladder marker.

40

2 h in 50 ~1 of an endogenous RT assay cocktail. The ingredients of the RT cocktail are standard, except for the addition of the random hexamer oligonucleotides. We found that the addition of this reagent increased the sensitivity of the RT-linked PCR procedures from lo- to lOO-fold (L. Donehower and R. Gibbs, unpublished observations). After the RT reaction was complete, a small aliquot (3 l.t.1)was subjected to the PCR procedure with the ‘universal’ primers (see Materials and Methods). Addition of more than 3 ul of RT reaction mix to the PCR mix was found to inhibit the PCR, possibly because of detergent in the RT cocktail. Ten t.~lof the PCR reaction were electrophoresed and visualized on an ethidium bromide stained agarose gel. Fig. 3 shows that the RSV-specific 135-bp fragment is generated and is cleaved at the appropriate sites by enzymes specific for the amplified RSV sequence. In an attempt to determine the sensitivity of the RT-linked PCR reactions, we performed serial dilutions of the purified RSV prior to the assay. The results indicated that only virus amounts above 10’ particles in the RT mix (or 5 x l@ particles in the PCR mix) consistently generate distinct RSV-specific 135-bp bands (data not shown). PURIFIED RETROVIRUS OR VIRAL RNA ENDDGENWS REVERSE TRANSCRIPTAX REACTIONOR cDNA SYNTHESIS

c

RETROVIRAL CoNAs PCR with “UNIVERSAL’ PRIMERS =

=

=

4

135 bp RETROVIRUS-SPECIFIC CNAFRAGMMS GEL PURIFICATION LIGATION OF 1.35bp FRAGMENT INTO BLUESCRIPT PHAGEMID

BLUESCRIF’T

c c

DIDEOXYSEQUENCINGOF INSERT USING TJAND 17 PRIMERS SEARCH OFGENBANK FOR SEQUENCE SIMIIARITIES

VIRUS IDENTIFICATION

Fig. 4. Schematic outline of the procedure used for identifying an uncharacterized

retrovirus.

41

Testing of the ‘universal’ retrovirus primer-based retrovirus

PCR procedure

on an unidentified

We next applied the ‘universal’ retrovirus primer-based PCR procedure to rapidly identify a retrovirus that we had recently isolated from a B-cell lymphoma of an AIDS patient (Bohannon et al., in preparation). This retrovirus was not related to HIV-l, HTLV-I, or HTLV-II by nucleic acid hybridization or antibody assays. Rather than perform extensive screenings with batteries of reagents from other nonhuman retroviruses or molecularly clone the viral genome (which can be slow and laborious and might be redundant-if the virus had already been cloned), we decided to use the ‘universal’ primer-based PCR method to identify this retrovirus. The procedures used to identify the uncharacterized virus are outlined in Fig. 4. Prior to PCR, a cDNA was generated from purified viral RNA utilizing a standard cDNA synthesis kit provided by Amersham. The cDNA derived was next amplified to generate a 135bp putative retrovirus-specific fragment. This fragment was gel-purified and blunt end ligated into the SmaI site of the BlueScript (Stratagene) plasmid after determining that the 135bp fragment was resistant to SmaI digestion. Recombinant plasmids were screened by restriction mapping and four plasmids with correct sized inserts were subjected to DNA sequencing, utilizing primers complementary to the T3 and T7 promoters adjacent to the polylinker cloning sites. Analysis of the sequence indicated that two of the four plasmid inserts were almost identical and had standard retroviral amino acid codon motifs (Fig. 5). Within the amplified 135-bp fragments, in addition to the highly conserved sequences from which the primers are derived, were two universally conserved amino acids, a serine and a proline residue four and five codons downstream, respectively, from the glytine codon in the 5’ primer (Fig. 5). In addition, the codons within the two primers were in the same reading frame and were separated by exactly 30 amino acids. This distance between primer codons is precisely conserved in all retroviruses sequenced to date. The other two sequences did not have these conserved motifs and appeared to be multimers of the original primers. When the fragments with retroviral like sequences were used to search the GenBank nucleotide sequence data base, the MPMV sequence (Sonigo et al., 1986) showed 96.6% similarity to the amplified probe sequence for clone 1 and 97.4% for clone 2 (Fig. 5). SRV-1 exhibited 91.5% and 92.3% similarity to the two clones. Most of the nucleotide differences between the clones and the MPMV sequence were within the primer sequences, which may reflect priming by mismatched oligonucleotides. The two non-primer differences between clone 1 and clone 2 may be a result of nucleotide misincorporation during amplification or nucleotide polymorphisms in the original viral RNA templates. The sequencing data indicate that the uncharacterized retrovirus may be MPMV or a related variant (e.g., SRV-1). Subsequent amino acid sequencing of the major viral core protein and immunological analyses confirmed that the isolated retrovirus was closely related to MPMV (Bohannon et al., unpublished). Further analyses are in progress.

2

CONSENSUS

Clone

v R c

P

a

G R

F N W

S

P

T A Y

F c Y

L V M

L I

Fig. 5. Comparison of the two sequenced amplified fragment clones with the corresponding MPMV sequence, SRV-1, and the retrovirus consensus sequence. The two sequenced clones are shown at the top, and both DNA sequence and derived amino acid sequences are indicated. The primer sequences are overlined. The dash within the clone 1 primer sequence indicates a single nucleotide deletion. The MPMV and SRV-1 nucleotide and ammo acid sequences are aligned below the clones. Nucleotide and amino acid differences with MPhW are underlined in the sequenced clones and SRV-1. Below the SRV1 sequence is the consensus amino acid sequence derived from 13 different retroviruses. Only positions with three or fewer different amino acid codons among all the viruses are indicated.

L

GT~TTACCACASGG~ATGGCCIV\CAGTC~ACC~ATGTC~TATGTGGCCACAGCCATACATMGG~AGACATGC~~~C~TGTATA~ATACA~ACATGGATGAC VLPQGWANSPTLCQKYVATAIHKVRHAWKQMYIIHY,,DD

Y

n V

D

D

43

Discussion The reverse transcriptases of all known retroviruses share short conserved sequences. Two of the most highly conserved regions are exactly the same distance apart in all viruses and can provide priming sites for amplification of a retroviral fragment (117 bp; 135 bp when non-complementary primer tails are included) (Mack and Sninsky, 1988; Shih et al., 1989). We have demonstrated that primers derived from these two conserved regions can amplify a variety of retroviral DNAs including DNA templates (e.g., MO-MLV) which are not perfectly complementary within a short 12 and 14 nucleotide length. Given the short length of primer complementarity, we have shown that the amplification reaction is reasonably specific. Only occasionally do the plasmids containing retroviral DNA generate secondary bands following PCR (Fig. 2). However, the specificity of the procedure is limited to genomes of low complexity, such as cloned DNAs and purified viral nucleic acids. The large number of retrovirus and retrovirus-like sequences in animal genomes precludes this as a virus identification procedure using unfractionated cellular nucleic acid temp!ates [unless the intent is to identify endogenous retroviral genomes specifically (Shih et al., 1989)]. The RT-linked PCR procedure utilizing the ‘universal’ primers was specifically designed to detect and identify potentially novel retroviruses rapidly. However, the retroviral template must be available outside the context of cellular nucleic acids. Therefore, this procedure should succeed if one can obtain moderate quantities (>lO’ particles) of well-purified retrovirus. At least two cycles of sucrose gradient purification are desirable. We have attempted to amplify retroviruses directly from cell culture medium and have found that 135bp fragments are obtained which are not virus-specific and are possibly derived from free cellular DNA in the culture medium. The sensitivity of the RT-linked PCR procedure is such that a virus-specific fragment can be generated with as few as 5 x lo5 retrovirus particles in the PCR mix (L. Donehower, unpublished data). This detection level is hardly impressive when compared to the amplification of DNA fragments from single-copy genes in a single cell (Li et al., 1988) and may be a result of the inefficiency in priming of the short degenerate oligonucleotides in the presence of virion reverse transcriptase. The conditions for optimal sensitivity with the ‘universal’ primers have not yet been determined. However, we have found that when perfect complementary primers of 25 nucleotides in length are employed in the RT-linked PCR reaction, as few as 100 viral particles can be routinely detected (L. Donehower and R. Gibbs, unpublished data). The RT-linked PCR procedure is relatively simple and can be completed in 6-8 h. If the appropriate size amplified fragment is obtained, it can be molecularly cloned and sequenced by routine procedures (Fig. 4). The fragment can be cloned into a vector with a blunt end restriction site or into a vector with BumHI and SuZI sites (following cleavage of the amplified fragment with these enzymes). If an uncharacterized retrovirus is being investigated, the ability to rapidly sequence a short amplified fragment of the virus may identify the retrovirus without the need to la-

44

boriously clone and subclone the viral genome by standard molecular cloning procedures. Even if the sequence information derived from the amplified fragment reveals that the retrovirus is not closely related to a known retrovirus, it can be classified into one of several retrovirus groups identified by Mack and Sninsky (1988) on the basis of conserved amino acid patterns. To test the ‘universal’ retrovirus primer-based PCR procedure, we utilized an unknown virus isolated from a B-cell lymphoma of an AIDS patient. In this case, we employed a variation on the RT-linked PCR procedure, utilizing cDNA synthesized from purified viral RNA (rather than virions) to amplify with the ‘universal’ primer-based PCR. This procedure has been adapted from Lee et al. (1988) who used degenerate primers to amplify a urate oxidase gene fragment from a cDNA library. We were able to obtain clones apparently derived from an amplified fragment of a retrovirus. Two of the four clones were almost identical in sequence and had conserved retrovirus motifs. The other two clones (which showed no apparent retrovirus conserved motifs) may have been the result of PCR artifacts. Unexpectedly, we found that the amplified fragments that were retroviral-like in sequence were, in fact, very similar to the MPMV and SRV-1, type D, immunosuppressive viruses which cause Simian AIDS in rhesus monkeys (Fine et al., 1975; Daniel et al., 1984; Marx et al., 1984; Stromberg et al., 1984). Because this virus was isolated from the patient’s tumor cells established in culture, our first suspicion was that it was a result of laboratory contamination. However, several considerations suggest the possibility that the patient may have been harboring this virus: (1) None of the labs involved in this study have ever worked with primate viruses; (2) the earliest in vitro passages of the tumor cells were expressing the retrovirus, whereas primary human B-lymphocytes cultured under the same conditions were not expressing the virus (which does efficiently infect human B cells); (3) the patient’s B cell tumors were an unusual syncytial variant (MPMV and SRV1 produce syncytia in some non-T human cell types (Fine et al., 1971; Daniel et al., 1984), while HIV-l primarily causes syncytia in CD4+ T cells); and (4) other reports in the literature indicate that humans may have been infected with these viruses or related relatives (Fine and Schochetman, 1978; Weiss, 1982). Further studies are in progress to determine whether the virus had infected the patient in vivo (Bohannon et al., unpublished data).

Acknowledgements We thank Dr Hamida Qavi and Dr David Steffen for the use of their DNA thermal cyclers, Steve Ressler for performing electron microscopic particle counts on the purified Rous sarcoma virus preparations, and Dr Wade Harper for help with the figures. This work was supported by grants CA09197, CA31479, and CA16672 from the National Cancer Institute.

45

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