Cellular gene expression survey of vaccinia virus infection of human HeLa cells - PubMed (original) (raw)

Cellular gene expression survey of vaccinia virus infection of human HeLa cells

Susana Guerra et al. J Virol. 2003 Jun.

Abstract

Vaccinia virus (VV) is a cytocidal virus that causes major changes in host cell machinery shortly after infecting cells. To define the consequences of virus infection on host gene expression, we used microarrays of approximately 15,000 human cDNAs to examine expression levels of mRNAs isolated at 2, 6, and 16 h postinfection from cultures of infected HeLa cells. The majority of profiling changes during VV infection corresponded to downregulation of genes at 16 h postinfection. Differentially expressed genes were clustered into seven groups to identify common regulatory pathways, with most of them (90%) belonging to clusters 6 and 7, which represent genes whose expression was repressed after infection. Cluster 1, however, contained 37 transcripts (2.81%) showing a robust pattern of induction that was maintained during the course of infection. Genes in cluster 1 included those for Wiskott-Aldrich syndrome protein (WASP) family member WASF1, thymosine, adenosine A2a receptor, glutamate decarboxylase 2, CD-80 antigen, KIAA0888 protein, selenophosphate synthetase, pericentrin, and attractin as well as several expressed sequence tags. We analyzed in more detail the fate of WASP protein in VV-infected cells, because a related family member, N-WASP, is involved in viral motility. WASP protein accumulated in the course of infection; its increase required viral DNA replication and de novo protein synthesis, and it localized in cytoplasmic structures distinct from uninfected cells. This study is the first quantitative analysis of host gene expression following VV infection of cultured human cells, demonstrating global changes in the expression profile, and identifies upregulated genes with potential roles in the virus replication cycle.

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Figures

FIG. 1.

FIG. 1.

Representation of 10 by 5 map obtained by the self-organizing maps algorithm, showing the gene expression clusters for VV-infected HeLa cells. Experimental points on the x axis are indicated as 1 for 2 h, 2 for 6 h, and 3 for 16 h postinfection. The y axis shows normalized expression values. Each cluster depicted was numbered from 1 to 7.

FIG. 2.

FIG. 2.

Characteristic expression patterns represented in each cluster. Mean values (left) and standard deviations (right) of the expression profiles of genes assigned to each cluster. Experimental points on the x axis are as in Fig. 1. The y axis shows normalized expression values. Cluster 4 was not included because it contains only one transcript (0.08%).

FIG.3.

FIG.3.

Validation of microarray data by Northern blot. Total RNA (20 μg) purified from uninfected and VV-infected cells at 2, 6, and 16 h postinfection was hybridized with probes derived from PCR products that were spotted on the microarray. The genes included in the autoradiogram are N34895 (EST), WASP (Wiskott-Aldrich syndrome protein), H2AFL (histone L), H2FB (histone B), EF (elongation factor), APEXL2 (apurinic/apyridiminic endonuclease), FLJ20643 (EST), and E3L (VV protein).

FIG. 4.

FIG. 4.

Changes in cytoskeletal components after VV infection. (A) Western blot comparison of actin and tubulin protein levels in lysates of mock- and VV-infected cells. Total proteins (100 μg) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-β-actin and anti-β-tubulin antibodies. (B) Densitometric quantification of tubulin protein. (C) Immunofluorescence analysis of the effect of VV infection on actin fibers in HeLa cells. Mock- and VV-infected cells at 6 and 16 h postinfection (hpi) were labeled with monoclonal antibody C3α14k to detect the A27L viral p14 protein, followed by the appropriate phalloidin-conjugated secondary antibody and ToPro reagent. The samples were analyzed by confocal immunofluorescence microscopy. (D) Immunofluorescence analysis of the effect of VV infection on tubulin.

FIG. 5.

FIG. 5.

Upregulation of WASP protein after VV infection. (A) Comparison of WASP protein levels by Western blot from lysates of mock- and VV-infected cells at 2, 6, and 16 h postinfection (hpi) and from cells treated at infection with cycloheximide (CHX; 100 μg/ml) and adenosine arabinoside (ARAC; 50 μg/ml) for 16 h. Total proteins (100 μg) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-WASP and anti-β-actin antibodies. (B) Densitometric quantification of WASP protein.

FIG. 6.

FIG. 6.

Immunofluorescence analysis showing WASP redistribution in VV-infected HeLa cells. Mock- and VV-infected cells at 6 and 16 h postinfection (hpi) were double-labeled with monoclonal antibody C3α14k to detect the A27L viral p14 protein and anti-WASP antibody, followed by the appropriate fluorescent secondary antibody and ToPro reagent. Cells were visualized by confocal immunofluorescence microscopy.

FIG. 7.

FIG. 7.

VV infection changes the subcellular localization of WASP from the Golgi complex. HeLa cells cultured on coverslips were infected with VV for 6 and 16 h. Cells were treated with anti-WASP antibody, followed by an appropriate fluorescent secondary antibody, ToPro, and fluorescent anti-wheat germ antigen (WGA).

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