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. 1999 Jul;73(7):5333-44.
doi: 10.1128/JVI.73.7.5333-5344.1999.

p53-Independent and -dependent requirements for E1B-55K in adenovirus type 5 replication

J N Harada  1 , A J Berk
Affiliations

p53-Independent and -dependent requirements for E1B-55K in adenovirus type 5 replication

J N Harada et al. J Virol. 1999 Jul.

Abstract

The adenovirus type 5 mutant dl1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53(-) and some p53(+) tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639-645, 1997). It has been suggested that dl1520 might be used as a "magic bullet" that could selectively lyse tumor cells without harm to normal tissues. However, we report here that dl1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce dl1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653-8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109-124, 1982), dl1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.

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Figures

FIG. 1
FIG. 1
p53 function is induced in H1299-tsp53 cells at 32°C. The p53 statuses of H1299, H1299-tsp53, and C33A cells were confirmed by examining p21 and hdm2 expression at temperatures previously determined to be nonpermissive (39°C) and permissive (32°C) for p53 A135V function (29, 57, 58). H1299, H1299-tsp53, and C33A cells were shifted from their normal growth temperature (39°C) to 32°C. At the indicated times after the temperature shift, equal numbers of cells were lysed in SDS sample buffer (73) and resolved by SDS–12% PAGE. Upon transfer to a nitrocellulose support, Western analyses of p21 and hdm2 were performed by using the WAF1 (Ab-1) and MDM2 (Ab-1) antibodies (Calbiochem). The positions of p21 and hdm2 are denoted by arrows, and molecular mass markers in kilodaltons (KD) are indicated at the right of each panel.
FIG. 2
FIG. 2
dl1520 replication is affected by both temperature and p53 function. Ad5 and dl1520 replication was monitored as a function of time postinfection in the H1299 and H1299-tsp53 cells at temperatures both nonpermissive (39°C) and permissive (32°C) for p53 A135V function. Infections at both temperatures were performed at an MOI of 5. Infections at 32°C were performed 24 h after the temperature shift from 39°C. Infected cells and supernatants were harvested at 24-h intervals after infection, and viral yields determined by assaying for plaque formation on 293 cells (33). Values are presented as the number of PFU derived per cell on a logarithmic scale and represent the averages from three independent experiments performed in duplicate.
FIG. 3
FIG. 3
Viral DNA replication is restricted by p53 in dl1520-infected cells. H1299 and H1299-tsp53 cells were mock infected or infected with wild-type Ad5 or dl1520 at an MOI of 5 as described in Materials and Methods. Infections were performed at both 39 and 32°C. Infections at 32°C were carried out 24 h after the shift from 39°C. Viral DNA was harvested from infected cells by a modified Hirt method (40) at the indicated times postinfection (p.i.). DNA isolated from an equal number of cells was digested with the restriction endonuclease XhoI and subsequently resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Known quantities of purified Ad5 DNA were included as standards for quantitation. The bands corresponding to the XhoI-digested viral DNA fragments are indicated by the brackets at right. The 1.0-kb plus ladder (Gibco BRL) is also included so that the sizes of the individual fragments may be delineated.
FIG. 4
FIG. 4
Effects of p53 on late RNA metabolism. H1299 and H1299-tsp53 cells were infected with Ad5 and dl1520 at an MOI of 30. Infections were performed at 32°C and at 24 h after the shift to the lower temperature. Nuclear (NUC) and cytoplasmic (CYT) RNAs were harvested at the indicated times postinfection (p.i.), and the accumulation of the fiber RNA was monitored by S1 analysis. Late fiber species were detected by utilizing a 32P-labelled oligonucleotide probe (Lifetech) overlapping the splice acceptor of the fiber gene. S1-protected products were resolved by urea-PAGE and subsequently visualized by PhosphorImager analysis. The 74- and 99-nucleotide (nt) protected species represent the mature and incompletely spliced forms of the fiber mRNA, respectively. Quantitation of the effects of p53 on nuclear and cytoplasmic RNA accumulation is shown in Table 2.
FIG. 5
FIG. 5
Protein expression during the late phase of infection in H1299 and H1299-tsp53 cells at 39 and 32°C. H1299 and H1299-tsp53 cells were infected with wild-type Ad5 or the E1B-55K mutant (dl1520) at 39 and 32°C. An MOI of 30 was used, and infections at 32°C were carried out at 24 h after the temperature shift. Late viral protein expression was monitored at various times postinfection (p.i.) by in vivo labeling with [35S]methionine-cysteine for a 1-h period. Lysates were TCA precipitated, and an equal number of precipitable counts was resolved by SDS–10% PAGE. The positions of the molecular mass markers are indicated in kilodaltons (KD) at the far right, and the bands corresponding to the major late proteins hexon (II), L4 100K protein, penton (III), and fiber (IV) are denoted by arrows on the left. The effects of temperature and p53 function on hexon and fiber synthesis were determined by PhosphorImager analysis and are shown in Table 2.
FIG. 6
FIG. 6
Adenovirus-induced cytopathic effect does not correlate with p53 genotype. Ad5- and dl1520-induced cytolysis was examined in the p53-positive WI38 (A to C) (26) and HNK (D to F) nonimmortalized cell strains and in the p53-negative SK-OV-3 (G to I) (85) and H1299 (J to L) (59) cell lines. Infections were performed at an MOI of 5 as described in Materials and Methods. Micrographs of infected cells were taken at 48 (B, E, H, and K) and 72 (C, F, I, and L) h postinfection (p.i.).
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References

    1. Arrigo S J, Chen I S Y. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991;5:808–819. - PubMed
    1. Babiss L E, Ginsberg H S. Adenovirus type 5 early region 1B gene product is required for efficient shutoff of host protein synthesis. J Virol. 1984;50:202–212. - PMC - PubMed
    1. Babiss L E, Ginsberg H S, Darnell J E. Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol Cell Biol. 1985;5:2552–2558. - PMC - PubMed
    1. Baker S J, Markowitz S, Fearon E R, Willson J K, Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990;249:912–915. - PubMed
    1. Barker D D, Berk A J. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology. 1988;56:107–121. - PubMed

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