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Journal of Virology, February 1999, p. 1245-1253, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Analysis of Synthesis, Stability, Phosphorylation,
and Interacting Polypeptides of the 34-Kilodalton Product of Open
Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus
Type 5
Dominique
Boivin,1,
Megan R.
Morrison,1
Richard C.
Marcellus,1,
Emmanuelle
Querido,1 and
Philip
E.
Branton1,2,*
Departments of
Biochemistry1 and
Oncology,2 McGill University,
Montréal, Québec, Canada H3G 1Y6
Received 9 March 1998/Accepted 27 October 1998
The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of
human adenovirus type 5 forms complexes with both the cellular tumor
suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can
inhibit p53 transactivation activity, as can E1B-55kDa, and in
combination these viral proteins cause the rapid turnover of p53. In
addition, E4orf6-55kDa complexes play a critical role at later times in
the regulation of viral mRNA transport and shutoff of host cell protein
synthesis. In the present study, we have further characterized some of
the biological properties of E4orf6. Analysis of extracts from infected
cells by Western blotting indicated that E4orf6, like E1A and E1B
products, is present at high levels until very late times, suggesting
that it is available to act throughout the infectious cycle. This
pattern is similar to that of E4orf4 but differs markedly from that of
another E4 product, E4orf6/7, which is present only transiently.
Synthesis of E4orf6 is maximal at early stages but ceases completely
with the onset of shutoff of host protein synthesis; however, it was
found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high
levels to be maintained even at late times. E4orf6 was shown to be
phosphorylated at low levels. Coimmunoprecipitation studies in cells
lacking p53 indicated that E4orf6 interacts with a number of other
proteins. Five of these were shown to be viral or virally induced
proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One
such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the
L4 100-kDa nonstructural adenovirus late product, but it appeared to be
present nonspecifically and not as part of an E4orf6 complex. Apart
from p53, three additional cellular proteins, of 84, 19, and 14 kDa
were detected by using an adenovirus vector that expresses only E4orf6.
The 19-kDa species and a 16-kDa cellular protein were also shown to
interact with E4orf6/7. It is possible that complex formation with
these viral and cellular proteins plays a role in one or more of the
biological activities associated with E4orf6 and E4orf6/7.
*
Corresponding author. Mailing address: Department of
Biochemistry, McGill University, McIntyre Medical Sciences Building, 3655 Drummond St., Montréal, Québec, Canada H3G 1Y6. Phone: (514) 398-8350. Fax: (514) 398-7384. E-mail:
branton{at}medcor.mcgill.ca

Present address: Geminx Biotechnologies,
Montréal, Québec, Canada H2W
2M9.
Journal of Virology, February 1999, p. 1245-1253, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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