A subset of viral transcripts packaged within human cytomegalovirus particles

Science

Volumn 288, Issue 5475, 2000, Pages 2373-2376

  Bresnahan, Wade A ^a   Shenk, Thomas ^a  

^a PRINCETON UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CONTROLLED STUDY; GENE EXPRESSION; HUMAN; HUMAN CELL; HUMAN CYTOMEGALOVIRUS; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; VIRION; VIRUS GENE; VIRUS GENOME; VIRUS PARTICLE; VIRUS REPLICATION; VIRUS TRANSCRIPTION;

CELL NUCLEUS; CELLS, CULTURED; CYTOMEGALOVIRUS; DACTINOMYCIN; GENE EXPRESSION; GENES, VIRAL; GENOME, VIRAL; GOLGI APPARATUS; HUMANS; NUCLEIC ACID HYBRIDIZATION; NUCLEIC ACID SYNTHESIS INHIBITORS; OLIGONUCLEOTIDE ARRAY SEQUENCE ANALYSIS; RECOMBINANT FUSION PROTEINS; RNA, MESSENGER; RNA, VIRAL; TRANSCRIPTION, GENETIC; VIRAL PROTEINS; VIRION; VIRUS ASSEMBLY;

EID: 0034733741     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5475.2373     Document Type: Article

References (27)

1. M. W. Wathen and M. F. Stinski, J. Virol. 41, 462 (1982).
2. H. Zhu and T. Shenk, unpublished data.
3. Primers were designed to amplify all 208 predicted HCMV ORFs of 100 amino acids or more (4, 23). The entire gene was amplified if it was equal to or Less than 600 nucleotides in length. For genes greater than 600 nucleotides, 300- to 400-base pair DNA fragments corresponding to the 3′ end of the gene were amplified. All DNA fragments were amplified by PCR with purified AD169 HCMV DNA as template. PCR products were gel purified and reamplified with the same primer pairs. DNA fragments were purified, and then concentrations were determined. DNA fragments were denatured in 0.8 M NaOH and 20 mM EDTA (pH 8.2), and 5 ng of each gene was spotted onto Nytran+ nitrocellulose membranes with a 386-pin multiblot replicator (VP-Scientific, San Diego, CA). DNA was ultraviolet-cross-Unked to the membranes. PCR products were cloned into the pGEMT-Easy vector (Promega, Madison, WI) so subsequent amplifications could be performed with a universal primer set.
4. M. S. Chee et al., Curr. Top. Microbiol. Immunol. 154, 125 (1990).
5. 32P)deoxycytidine triphosphate to generate a radioactively labeled cDNA.
6. W. D. Rawlinson and B. G. Barrell, J. Virol. 67, 5502 (1993).
7. UL21.5 transcription is greatly diminished in the presence of the viral DNA polymerase inhibitor phosphonoacetic acid, classifying the UL21.5 transcript as an early-late transcript (15).
8. B. Platcher, B. Traupe, J. Albrecht, G. Jahn, J. Gen. Virol. 69, 2251 (1988).
9. S. H. McDonough, S. I. Strapens, D. H. Spector, J. Virol. 53, 711 (1985).
10. N. I. Hutchinson, R. T. Sondermeyer, M. J. Tocci, Virology 155, 160 (1986).
11. M. F. Stinski, J. Virol. 19, 594 (1976).
12. I. Sarov and I. Abady, Virology 66, 464 (1975).
13. A. Irmiere and W. Gibson, Virology 130, 118 (1983).
14. Virions were sequentially pelleted through a sorbitol cushion, sedimented in a glycerol-tartarate gradient (24), treated with RNase One, and centrifuged to equilibrium in a CsCl gradient (11).
15. W. A. Bresnahan and T. Shenk, data not shown.
16. J. Mullberg et al., J. Gen. Virol. 80, 437 (1999).
17. The recombinant adenovirus was generated as described previously (25). Briefly, UL21.5 was amplified by PCR from the AD169 genome (nucleotides 27,108 to 27,499). The PCR fragment was sequenced and subcloned into pEYFP (Clontech, Palo Alto, CA) to generate pUL21.5-YFP through a Hind III-Sac II digest. The UL21.5-YFP coding sequence was excised with BgI II-Not I and cloned into pAdShuttle (25). Homologous recombination of Prne I-linerized pAdShuttle 21.5-YFP DNA with the viral backbone vector pAd-Easy1 (25) was performed in Escherichia coli BJ5183 cells, yielding a replication-deficient adenovirus lacking the E1 and E3 transcription units (pAdShuttle UL21.5-YFP). Virus was cultured in 293 cells and isolated and purified as described (25).
18. M. G. Waters, D. O. Clary, J. E. Rothman, J. Cell. Biol. 118, 1015 (1992).
19. Recombinant HCMV expressing UL21.5-YFP in place of wild-type UL21.5 was generated by PCR amplification of the UL21.5 5′ (25,921 to 27,499) and 3′ (nt 27,503 to 29,096) flanks with purified AD169 genomic DNA as template. The 5′ flank (containing UL21.5 coding sequence) was subcloned into pEYFP through a Hind III-Sac II digest generating pUL21.5-YFP 5′. The 3′ flank was amplified and cloned into pGEMT-Easy (Promega, Madison, WI) and subsequently subcloned into pUL21.5-YFP 5′ through Not I digestion generating pAD169UL21.5-YFP. Vector sequences were removed from pAD169UL21.5-YFP by Nru I digestion. Recombinant HCMV virus was generated through homologous recombination within transfected human fibroblasts as described (26).
20. R. J. Roller and B. Roizman, J. Virol. 64, 3463 (1990).
21. _, J. Virol. 66, 3624 (1992).
22. M. B. Mathews and T. Shenk, J. Virol. 65, 5657 (1991).
23. A complete list of primers used is available at www. sciencemag.org/feature/data/1050215.shl.
24. C. J. Baldick and T. Shenk, J. Virol. 70, 6097 (1996).
25. T.-C. He et al., Proc. Natl. Acad. Sci. U.S.A. 95, 2509 (1998).
26. A. J. Hirsch and T. Shenk, J. Virol. 73, 404 (1999).
27. We thank L. Enquist, D. Yu, and F. Goodrum for critical reading of the manuscript; B. Vogelstein for the Ad-Easy-Adenovirus expression System; G. Hultman for technical assistance; and H Zhu for sharing unpublished results. Supported by a grant from the NIH (CA85786). W.A.B. was supported by a Jeane B. Kempner postdoctoral fellowship.