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. 2006 Jan;80(2):964-74.
doi: 10.1128/JVI.80.2.964-974.2006.

Adenovirus E1B 55-kilodalton protein is required for both regulation of mRNA export and efficient entry into the late phase of infection in normal human fibroblasts

Ramon Gonzalez  1 , Wenying Huang, Renee Finnen, Courtney Bragg, S J Flint
Affiliations

Adenovirus E1B 55-kilodalton protein is required for both regulation of mRNA export and efficient entry into the late phase of infection in normal human fibroblasts

Ramon Gonzalez et al. J Virol. 2006 Jan.

Abstract

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).

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Figures

FIG. 1.
FIG. 1.
The kinetics of Ad5 replication in HeLa cells and HFFs. Following infection at 0.1 PFU/cell, cells were harvested at regular intervals, and yields of infectious particles were determined by duplicate plaque assays of multiple dilutions of each sample. p.i., postinfection.
FIG. 2.
FIG. 2.
The kinetics of synthesis of viral DNA and early proteins in Ad5-infected HFFs. A. The concentrations of viral DNA present in HFFs infected with 30 PFU/cell Ad5 for the periods indicated were determined by blotting and hybridization to viral DNA as described in Materials and Methods. Signals were quantified and corrected using rRNA genes as an internal control and are expressed relative to the maximal quantity of viral DNA detected. The values shown represent means for two independent experiments. B. Total cell extracts prepared from HFFs infected with 30 PFU/cell Ad5 for the periods indicated were assayed for the presence of the E1A and E1B 55-kDa proteins by using immunoblotting as described in Materials and Methods. p.i., postinfection.
FIG. 3.
FIG. 3.
Effects of insertion mutations on accumulation of the E1B 55-kDa protein. A. The 496-residue Ad5 E1B 55-kDa protein is represented by the rectangle at the top, on which are shown the positions of the leucine-rich export signal (NES), an RNP RNA-binding motif (RNP), and a putative C2H2 zinc finger (Zn finger). The sites at which the protein is modified are indicated above the protein. The sites of the four-amino-acid insertions carried by the mutants used in these experiments are indicated below the protein. The effects of these mutations on binding of the E1B 55-kDa protein to other proteins (34, 55, 83) and on viral late mRNA export in HeLa cells (39) are summarized below the insertion sites, where - and - indicate inhibition of the activity or function listed at the left. B. HFFs were infected for 20 or 40 h with 30 PFU/cell Ad5 (lanes 1 and 6), A143 (lanes 2 and 7), H224 (lanes 3 and 8), or H354 (lanes 4 and 9) or mock infected (lanes 5 and 10). The E1B 55-kDa protein was examined by immunoblotting, as described in Materials and Methods.
FIG. 4.
FIG. 4.
Synthesis of viral late proteins in HFFs infected by E1B mutant viruses. A. HFFs infected with 30 PFU/cell of the viruses indicated at the top or mock infected (M) were labeled with [35S]methionine plus [35S]cysteine during the late phase of infection, and cytoplasmic proteins were examined by SDS-polyacrylamide gel electrophoresis and autoradiography. The positions to which prestained molecular mass markers migrated are listed at the left, and those of prominent viral late proteins are indicated at the right. B. The steady-state concentrations attained by protein V 30 h after infection of HFFs with the viruses indicated at the top or in mock-infected cells (M) were compared by immunoblotting as described in Materials and Methods.
FIG. 5.
FIG. 5.
Effects of E1B mutations on the production and export of L2 fiber mRNA in HFFs. The concentrations of mature fiber mRNA present in nuclear and cytoplasmic fractions of HFFs infected for 30 to 32 h with 30 PFU/cell of the viruses indicated were determined by real-time RT-PCR as described in Materials and Methods. The nuclear concentrations and cytoplasmic/nuclear (C:N) concentration ratios, a measure of export efficiency, are expressed relative to the values observed for Ad5-infected cells. The values represent the means for three independent experiments.
FIG. 6.
FIG. 6.
Effects of the A143 mutation on the intracellular location of the E1B 55-kDa protein. HFFs infected with 30 PFU/cell of Ad5 or A143 or mock infected, as indicated, were processed for immunofluorescence 30 h after infection, and the locations of the viral E1B 55-kDa and E2 72-kDa proteins were examined as described in Materials and Methods. Panels i and n show merged images of Ad5- and A143-infected cells, respectively, at higher magnification. White arrows show colocalization of the two viral proteins.
FIG. 7.
FIG. 7.
Effects of E1B mutations on viral DNA synthesis in HFFs. Viral DNA was purified from HFFs infected for 30 h with the viruses indicated, loaded onto nylon membranes, and hybridized to labeled Ad5 DNA sequences as described in Materials and Methods. In the example shown in panel A, hybridization was to L3 viral DNA. Quantification of several such blots using rRNA genes as the internal control is shown by the gray bars in panel B. The variance shown for Ad5 was obtained by arbitrarily setting one of the signals as 1.0 and calculating the others relative to it. The black bars summarize the results obtained from three separate infections.
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