Differential distribution of the adenovirus E1A proteins and colocalization of E1A with the 70-kilodalton cellular heat shock protein in infected cells

Abstract

Five distinct localization patterns were observed for the adenovirus ElA proteins in the nuclei of infected HeLa cells: diffuse, reticular, nucleolar, punctate, and peripheral. The variable distribution of ElA was correlated with the time postinfection and the cell cycle stage of the host cell at the time of infection. All staining patterns, with the exception of peripheral ElA localization, were associated with the early phase of infection since only the diffuse, reticular, nucleolar, and punctate staining patterns were observed in the presence of hydroxyurea. Because the ElA proteins (12S and 13S) stimulate the expression of the cellular heat shock 70-kilodalton protein (hsp7O), we examined the intracellular distribution of hsp7O in the adenovirus-infected cells. Whereas hsp7O was predominantly cytoplasmic in the cells before infection, after adenovirus infection most of the protein was now found within the nucleus. Specifically, hsp7O was found within the nucleoli as well as exhibiting reticular, diffuse, and punctate nuclear staining patterns, analogous to those observed for the ElA proteins. Double-label indirect immunofluorescence of ElA and hsp7O in infected cells demonstrated a colocalization of these proteins in the nucleus. Translocation of hsp7O to the nucleus was dependent upon both adenovirus infection and expression of the ElA proteins. The localization of hsp7O was unaltered by infection with an ElA 9S cDNA virus which does not synthesize a functional ElA gene product. Moreover, the discrete nuclear localization patterns of ElA and the colocalization of ElA with hsp7O were not observed in adenovirus-transformed 293 cells which constitutively express ElA and ElB. ElA displayed exclusively diffuse nuclear staining in 293 cells; however, localization of ElA into the discrete nuclear patterns occurred after adenovirus infection of 293 cells. Immunoprecipitation of labeled infected-cell extracts with a monoclonal antibody directed against the ElA proteins resulted in precipitation of small amounts of hsp7O along with ElA. These data indicate that the adenovirus ElA proteins colocalize with, and possibly form a physical complex with, celHular hsp7O in infected cells. The relevance of this association, with respect to the function of these proteins during infection and the association of other oncoproteins with hsp7O, is discussed.

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding.

“…Recent biochemical evidence suggests that HSP70 and P72 are involved in the translocation of nascent polypeptides across membranes of the endoplasmic reticulum and mitochondria (7,12,28,47). HSP70 and P72 have also been shown to interact with clathrin, the cellular protooncogene p53, and the viral trans-activating proteins of adenovirus (E1A) and SV40 (T antigen) (4,5,13,34,37,38,44). Finally, we have demonstrated that HSP70 displays a number of cell cycle-specific interactions with other proteins (23).…”

An Hsp60 related protein is associated with purified HIV and SIV

“…This has led to speculations that Hsp's may play specific roles in viral replication. Furthermore, Hsp's have been reported to associate with some viral proteins [16,22,23,35] and in some instances with the mature virion [30]. However, the induction of Hsp's by viral infection may target that cell for elimination by the immune system [18,24].…”

The Retinoblastoma Protein: A Master Regulator of Cell Cycle, Differentiation and Apoptosis

“…The developing mouse lens turned out to be a suitable model system to study the role of pRb in apoptosis and, beyond that, its cooperativity with p53. In this model system viral oncoproteins were used to specifically neutralise pRb such as SV40 Tag (DeCaprio et al, 1988), adenoviral E1A (White et al, 1988) and HPV E7 (Dyson et al, 1989;Munger et al, 1989), and p53 functions [SV40 Tag (Lane and Crawford, 1979;Linzer and Levine, 1979); 5.5-kD E1B (Sarnow et al, 1982); HPV E6 (Werness et al, 1990;Huibregtse et al, 1993)] thus mimicking the loss of either of the two proteins. Binding of these viral proteins, on one side, disrupts the association between pRb and E2F thus allowing the activation of downstream target genes by E2F (Nevins, 1992), on the other side, it interferes with the ability of p53 to eliminate inappropriately growing cells by triggering apoptosis (Clarke et al, 1993;Lowe et al, 1993).…”

“…The mechanism by which pRb acts as a tumour suppressor and its function in the normal cell cycle control originates from investigations unraveling the mechanism of how oncoviral proteins function to deregulate cell cycle control. The SV40 Tag (De Capno et al, 1988), the adenovirus E1A protein (White et al, 1988), and the human papilloma virus-16 (HPV) protein E7 (Dyson et al, 1989;Munger et al, 1989) all interact with the hypophosphorylated form of pRb and its relatives p107 and p130 by binding to their A/B pocket domains Cao et al, 1992;Devoto et al, 1992;Lees et al, 1992;Shirodkar et al, 1992). The finding that the same sequence motif in the viral proteins (LXCXE) is responsible for (a) the oncogenic capacity of the virus, (b) tethering pRb and (c) releasing the transcription factor E2F from a complex with pRb (Bagchi et al, 1991 ;Chellappan et al, 1991Chellappan et al, , 1992Chittenden et al, 1991) has led to the now widely accepted model that viral oncoproteins promote growth by sequestering pRb which normally acts to suppress growth by binding E2F and thus hinder the activation of downstream targets needed for S phase entry (reviewed by Nevins, 1992;LaThangue, 1994;Vousden, 1995).…”