Nucleic Acids Research, Vol. 18, No. 13

4029

A replication-deficient HIV-1 DNA used for quantitation of the polymerase chain reaction (PCR) Clyde Hart*, Shen-Yung Chang', Shirley Kwokl, John Sninsky', Chin-Yih Ou and Gerald Schochetman Centers for Disease Control, Atlanta, GA 30333 and 'Cetus Corporation, Emeryville, CA 94608, USA Submitted June 5, 1990

Quantitation of human immunodeficiency virus type 1 (HIV-1) proviral DNA copy number by PCR requires the use of known amounts of a plasmid containing HIV-1 DNA. A plasmid containing the entire proviral genome would allow any region to be targeted for amplification. To ensure the biosafe use of a full-length proviral DNA, a replication-deficient HIV-1 proviral DNA was developed. Previous reports have shown the requirement for a functioning HIV-1 protease gene in core protein maturation and viral infectivity (1-3). To construct an HIV-1 DNA standard containing a modified protease gene, the pZ6Neo plasmid containing the infectious molecular clone HIVZ6 (GenBank accession no. K03458) was cleaved with restriction endonuclease SacI. The resulting 9 kb fragment containing the complete HIV- 1 genome was circularized, cleaved with restriction endonuclease BclI (position 2431 of HIVZ6), and incorporated between the BamHI and NruI restriction endonuclease sites of PBR322. This construct (pSYC1857) contained an interruption of the pol gene of HIVZ6 in the protease coding region (Figure 1). Virus production was assayed in human rhabdomyosarcoma (RD) cells by DNA transfection of pSYC1857, BglI-digested pSYC1857 or pZ6Neo. Pelletable (105xg) HIV p24 antigen was greater than 250 pg/ml in culture media of RD cells transfected with all three constructs. Reverse transcriptase (RT)

1-

activity in these cultures was detected only from pZ6Neo transfected cells (1.1 x 105 cpm of [3H]TTP incorporation/ml of culture media). To determine if infectious virus was produced by the transfected RD cells, Hut78 cells were inoculated with the culture supernatants of the transfected cells. Only Hut78 cultures inoculated with supernatant from pZ6Neo transfected cells had detectable RT activity (Figure 2) and pelletable HIV p24 antigen. PCR analysis showed that the pSYC 1857 construct was detectable down to 1 copy of DNA per 1 x 105 cells (Figure 3). These observations demonstrate that pSYC 1857 DNA does not support production of detectable infectious virus and will be useful as a standard for quantitation of HIV-1 DNA by PCR.

REFERENCES 1. Gottlinger,H.G., Sodroski,J.G. and Haseltine,W.A. (1989) Proc. Natl. Acad. Sci. USA 86, 5781-5785. 2. Kohl,N.E. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 4686-4690. 3. Peng,C., Ho,B.K., Chang,T.W. and Chang,N.T. (1989) J. Virol. 63, 2550-2556. 4. Kellogg,D.E. and Kwok,S. (1990) In PCR Protocols: A guide to methods and applications. Innis,M.A., Delfand,D.H., Sninsky,J.J. and White,T.J. (eds). 337-347.

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Figure 1. Construction of the pSYC1857 plasmid. pZ6Neo plasmid DNA was cut with the restriction enzyme SacI. The resulting 9 kb fragment containing the HIV genome was circularized, cut with restriction enzyme BclI, and ligated between the BamHI and NruI restriction sites of PBR322.

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A replication-deficient HIV-1 DNA used for quantitation of the polymerase chain reaction (PCR).

Nucleic Acids Research, Vol. 18, No. 13 4029 A replication-deficient HIV-1 DNA used for quantitation of the polymerase chain reaction (PCR) Clyde Ha...
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