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. viduals (8, 58). Virtually all cell types, with the exception of lymphocytes and polymorphonuclear leukocytes, can support CMV replication in vivo (80), and this remarkably broad tropism is at the basis of the numerous clinical manifestations of CMV infection (8, 58). The range of permissive cells in vitro is more limited, with human fibroblasts (HF) and endothelial cells being the most widely used for propagation of clinical isolates. Two extensively studied strains, AD169 and Towne, were generated by serial passage of tissue isolates in HF for the purpose of vaccine development (22,68). During this process, both strains accumulated numerous genomic changes (11) and lost the ability to grow in cell types other than HF. By contrast, propagation in endothelial cells produced strains with more intact genomes and tropism, such as TB40/E, VR1814, TR, and PH (59, 80). Human cytomegalovirus (CMV) is a ubiquitous herpesvirus that can cause serious disease in immunocompromised indiThe viral determinants of endothelial and epithelial cell tropism have recently been mapped to the UL128-UL131A (UL128-131A) genomic locus (32,92,93). Each of the products of the UL128, UL130, and UL131A genes is independently required for tropism and participates in the formation of a complex at the surface of the virion with the viral glycoproteins gH and gL (74, 93), which can also independently associate with gO (45). The gH/gL/UL128-131A complex appears to be required for entry into endothelial cells by endocytosis, followed by low-pH-dependent fusion of the virus envelope with endosomal membranes (73, 74) although some virus strains expressing the UL128-UL131A genes do not require endosome acid...

The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus

“…using an Acumen e X3 cytometer to determine % infection. We observed a difference in the EC 50 value of podofilox against TB40/E WT (Figure 6 and Table 1) that could be due to TB40/E’s reliance on vimentin and intermediate filaments in contrast to AD169 [42]. What was most remarkable was the differences in the maximal plateau inhibition (MPI) [43] of podofilox based on Prism5’s non-linear regression (Figure 6 and Table 1).…”

Human Cytomegalovirus Infection of Langerhans-Type Dendritic Cells Does Not Require the Presence of the gH/gL/UL128-131A Complex and Is Blocked after Nuclear Deposition of Viral Genomes in Immature Cells

“…To identify the step in the viral cycle that is blocked in iLC, the progress of viral particles was tracked by staining infected iLC and mLC for pp150, a tegument phosphoprotein that remains strongly associated with capsids during entry and has been used as a capsid marker by us and others (60,74,75). Cytospin preparations of iLC and mLC exposed to BAC4 at an MOI of 10 PFU/cell and harvested at 0.25, 1, 5, 20, and 30 hpi were fixed and permeabilized prior to the addition of anti-pp150 antibodies.…”

Dynamics of Human Cytomegalovirus Infection in CD34+Hematopoietic Cells and Derived Langerhans-Type Dendritic Cells

“…Population-wise, LC are much less susceptible to CMV infection than HFF. When infected at an MOI of 10, only about 2% Ϯ 0.7% of iLC and 10% Ϯ 5% of mLC (9), but between 80% and 100% of HFF (65), express the IE1/IE2 proteins. While the percentage of IE1/IE2 ϩ HFF can be increased by raising the MOI, the total numbers of LC expressing the IE1/IE2 proteins after infection at MOIs of 10 and 100 remain virtually identical.…”