Onset of human cytomegalovirus replication in fibroblasts requires the presence of an intact vimentin cytoskeleton

Abstract

Like all viruses, herpesviruses extensively interact with the host cytoskeleton during entry. While microtubules and microfilaments appear to facilitate viral capsid transport toward the nucleus, evidence for a role of intermediate filaments in herpesvirus entry is lacking. Here, we examined the function of vimentin intermediate filaments in fibroblasts during the initial phase of infection of two genotypically distinct strains of human cytomegalovirus (CMV), one with narrow (AD169) and one with broad (TB40/E) cell tropism. Chemical disruption of the vimentin network with acrylamide, intermediate filament bundling in cells from a patient with giant axonal neuropathy, and absence of vimentin in fibroblasts from vimentin(-/-) mice severely reduced entry of either strain. In vimentin null cells, viral particles remained in the cytoplasm longer than in vimentin(+/+) cells. TB40/E infection was consistently slower than that of AD169 and was more negatively affected by the disruption or absence of vimentin. These findings demonstrate that an intact vimentin network is required for CMV infection onset, that intermediate filaments may function during viral entry to facilitate capsid trafficking and/or docking to the nuclear envelope, and that maintenance of a broader cell tropism is associated with a higher degree of dependence on the vimentin cytoskeleton.

FIG. 1.

Organization of the vimentin cytoskeleton during entry of AD169 in HF. Mock-infected (A to D) or AD169-infected (MOI of 5; E to X) HF harvested at 5 or 30 min postinfection and at 1, 2, or 4 hpi were stained with a monoclonal antivimentin antibody followed by an Alexa Fluor 594-conjugated goat anti-mouse antibody (red) and a fluorescein isothiocyanate-conjugated anti-IE1/IE2 antibody (green). Nuclear DNA was highlighted with Hoechst 33342 (blue). Merged images are shown in panels on the right. Original magnification was ×40 for panels A to D and ×100 for panels E to X.

FIG. 2.

AD169 and TB40/E infection time course in HF. Cells and supernatants from cultures infected with AD169 or TB40/E at an MOI of 1 (A and C) or 5 (B) were collected at the indicated times postinfection. (A and B) Cells were stained for IE1/IE2, and the percentage of expressing cells was calculated. Means and standard deviations of the percentage values of IE1/IE2-positive cells scored in five separate fields per sample in one representative experiment are shown. (C) Amount of cell-free virus released in supernatants of AD169- and TB40/E-infected HF as quantified by plaque assay.

FIG. 3.

Effects of ACR pretreatment and AD169 infection on IF organization and nuclear morphology in HF. (A to F) Fluorescence microscopy analysis of vimentin IF organization in untreated HF (A) and in HF exposed to a 5 mM solution of ACR for 2 (B), 4 (C), 6 (D), or 8 h (E) prior to staining with antivimentin antibodies. (F) Confocal microscopy image of lamin B-stained cell nuclei after 6 h of ACR pretreatment. (G and H) Percentage of cells with nuclear invaginations (G) or with nuclear outlines consistent with apoptosis (H) in HF monolayers harvested immediately after ACR treatment for the indicated times (white bars) or harvested after ACR removal and incubation of cells in fresh medium for 4 h (black bars). (I and J) Percentage of cells with nuclear invaginations (I) or with apoptotic nuclei (J) in HF monolayers harvested after ACR treatment for the indicated times and AD169 infection at an MOI of 1 (white bars) or 5 (black bars) for 4 h. Mean and standard deviation values from three independent experiments are shown.

FIG. 4.

Impact of ACR pretreatment on AD169 and TB40/E infection efficiency. (A and B) Percentage of IE1/IE2-expressing HF either untreated (No ACR) or after exposure to a 5 mM solution of ACR for 2, 4, 6, or 8 h prior to infection with AD169 or TB40/E at an MOI of 1 or 5 for 4 h. (C and D) Percentage of IE1/IE2-expressing HF left untreated (white bars) or treated with a 5 mM solution of ACR for 4 h (black bars) prior to infection with AD169 or TB40/E at an MOI of 1. Mean and standard deviation values from three (A and C) and two (B and D) independent experiments are shown.

FIG. 5.

Structure of vimentin IF and expression of viral IE1/IE2 proteins in WG0321 and WG0321 dermal fibroblasts. (A to F) Serum-starved dermal fibroblasts from healthy donors (MCH070) and from patients with GAN (WG0321) were stained for vimentin and with Hoechst 33342. Merged images are shown as indicated. (G and H) Serum-starved MCH070 (G) and WG0321 (H) dermal fibroblasts were infected with AD169 at an MOI of 10 for 4 h prior to staining for vimentin (green) and for IE1/IE2 (red). (I to L) Percentage of serum-starved MCH070 and WG0321 cells expressing IE1/IE2 after infection with AD169 (I and K) or TB40/E (J and L) at an MOI of 1 or 10. Mean and standard deviation values from three (I, K, and L) and two (J) independent experiments are shown. ND, not detected.

FIG. 6.

Viral IE1/IE2 gene expression in vim⁺ and vim⁻MEF. Percentage of vim⁺ (white bars) and vim⁻ (black bars) MEF expressing IE1/IE2 after infection with AD169 (A and C) or TB40/E (B and D) at an MOI of 1 or 10. Mean and standard deviation values from two independent experiments are shown.

FIG. 7.

Detection of virus particles in vim⁺ and vim⁻ MEF. (A to D) Confocal immunofluorescence images of AD169-infected MEF stained for pp150 at 1 and 8 hpi. The pp150 signal is depicted in red while the green signal emanates from cellular autofluorescence. (E) Percentage of AD169- and TB40/E-infected vim⁺ and vim⁻ MEF containing pp150- positive particles immediately after adsorption (black bars) and at three different times postpenetration (dark gray, light gray, and white bars; times are indicated at the top of the panel). A minimum of 110 cells were counted for each sample. Mean and standard deviation values from separate cell fields in one representative experiment (out of three) are shown. (F) Proportion of pp150-positive particles localizing at the cell surface, in the cytoplasm, or at the nucleus in vim⁺ and vim⁻ MEF infected with AD169 or TB40/E at 1 h postpenetration. Mean and standard deviation of values from eight different cells per sample are shown. AD, AD169; TB, TB40/E.

FIG. 8.

Hypothetical steps during CMV entry requiring vimentin assistance for efficient completion. Vimentin IF and microtubules are shown as three parallel thin lines and as thick gray lines, respectively. Black hexagons enclosed in a circle depict enveloped virions while isolated black hexagons represent virus capsids. Steps are as follows: (a) vimentin IF acting as receptors for CMV virions at the surface, (b) AP-3-mediated involvement of vimentin IF in internalization of clathrin-coated endosomes, (c) enhancement of capsid attachment and movement along microtubules via vimentin IF, (d) internalization of integrin-bound virions under the control of vimentin IF, (e) AP-3-mediated involvement of vimentin IF in endosome acidification and cytoplasmic release of capsids, and (f) facilitation of nuclear genome deposition and of gene transcription onset by nuclear lamina- and matrix-associated vimentin. MTOC, microtubule organizing center.