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Total cytoplasmic RNA isolated from uninfected or infected HEL fibroblast cells was subjected to RTPCR after treatment with deoxyribonuclease I. The first-strand complementary DNA was synthesized by Superscript II reverse transcriptase (Gibco/BRL) with antisense primer PR2 (5′-CATGGGTACCGTTGCCCCTGAATCCCAG-3′) derived from the 5′ region of pol (nucleotides 2405 to 2422; GenBank accession number M19427). Reaction products were subjected to PCR amplification with primers R+3 (5′-GCATCCCGGGGCTCTTCACTACTCGCTGCGT-3′) (nucleotides 3 to 23) and PR2, and products were analyzed. A major ∼600-bp fragment was isolated from the gel and cloned to generate pSGC5, and the DNA sequence determined
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7 virions, we concluded that there is approximately one DNA molecule present in every six virions In an independent experiment we found a DNA molecule in every nine virions In viral preparations harvested after 48 hours, we determined that 1 in 25 to 100 supernatant virions is infectious as measured in the FAB assay.
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We thank M. Groudine and M. Emerman for their antiques of the manuscript and A. Rethwilm for stimulating discussions about the HFV genome. We also thank A. R and R. Flugel for providing antisera and L. Caldwell and the electron microscopy facility at the Fred Hutchinson Cancer Research Center for particle counts. Supported by NIH grants CA18282 and HL53762 to M.L.L S.F.Y. was partially supported by a postdoctoral fellowship (F32 CA60357) from the National Cancer Institute.
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