Efficient Lytic Infection of Human Arterial Endothelial Cells by Human Cytomegalovirus Strains

Abstract

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) inEndothelial cells (EC) are major targets of human cytomegalovirus (HCMV) during acute infection of an immunocompromised host (18). In addition to contributing to hematogenous viral dissemination (7,8,13,19,23), infected EC may trigger direct vascular injury if viral induced cytopathogenicity occurs. In support of these in vivo data, susceptibility of cultured human venous EC (HUVEC) to productive lytic HCMV infection has been demonstrated (25). However, studies of interactions of HCMV with human arterial EC (HAEC) have generated conflicting results. Whereas reports about noncytopathic persistence of HCMV in cultured aortic EC (6) raised the possibility of different susceptibilities of EC depending on their vascular origin, Knight et al. (9) have described similar cytopathologies of HCMV in HUVEC, HAEC, and microvascular EC with regard to adhesion molecule induction. The respective usage of fibroblast-propagated and EC-propagated HCMV strains might provide an explanation for the discrepancies. While such interstrain differences in the cytopathic potential of HCMV in HUVEC are well established (11,20,22,24), the lytic potentials of various HCMV strains in HAEC have not yet been determined.In the present investigation we have tested the hypothesis that interstrain differences in viral host cell tropism, rather than properties inherent to EC of different vascular origins, determine the outcome of HCMV infection. To this end we have performed a detailed analysis of viral replication kinetics, long-term evolution of cytopathology, and lytic end points, comparing EC derived from human adult iliac artery, placental microvasculature, and umbilical vein following inoculation with various HCMV strains. Efficient lytic infection of HAEC by EC-propagated HCMV strains.In a first set of experiments, HCMV strains VHL/E (kindly provided by W. J. Waldman) (24) and TB40/E (22) were tested for cytopathic potential in HAEC. Both strains were propagated in HUVEC to preserve the natural endothelial cytopathogenicity of the original isolates. To obtain high-titer virus preparations, EC-propagated virus stocks were inoculated with fibroblasts for a single round of infection, harvested at 100% cytopathic effect (CPE), and made cell free by centrifugation. HAEC were isolated from adult human iliac arteries of bypass recipients by mechanically removing the endothelial layer as previously described (1, 2) and were cultured in EGM-2 medium (Clonetics, Walkersville, Md.) containing 2% fetal calf serum on culture flasks (Greiner, Frickenhausen, Germany) coated with 2% gelatin. For infection, cells were preincubated for 2 h in heparin-free medium, inoculated with cell-free virus at multiplicities of infection (MOI) of 0.1, 1, and 10, and incubated for 90 min before removal of the inoculum. The medium was replaced at 48-h intervals. The kinetics of viral antigen expression in HAEC cultures was analyzed by indirect immunoperoxidase detection of imm...

Rescue of human cytomegalovirus strain AD169 tropism for both leukocytes and human endothelial cells

“…However, strain AD169 may infect and replicate to some extent in aortic endothelial cells (Fish et al, 1998;BolovanFritts & Wiedeman, 2001), where virus infection does not spread to contiguous cells, resulting in a nonlytic persistent infection (Fish et al, 1998). Opposite results have been reported by others showing that arterial EC may undergo a lytic infection if an EC-adapted HCMV isolate is tested (Kahl et al, 2000). Our results show that even strain AD169 is cytopathogenic for both venous and arterial EC if it has been adapted to growth in this cell system.…”

“…Thus, EC-tropism involves virus adsorption, penetration, transport through the cytoplasm and then viral DNA access to the nucleus to trigger immediate-early transcriptional processes. AD169 has been repeatedly reported not to grow in HUVEC (MacCormac & Grundy, 1999;Sinzger et al, 2000) and the underlying mechanism has been attributed to the impairment or inefficiency of translocation of the viral genome into the nucleus of infected cells (Slobbe van Drunen et al, 1998;Sinzger et al, 2000;Bolovan-Fritts & Wiedeman, 2002). However, strain AD169 may infect and replicate to some extent in aortic endothelial cells (Fish et al, 1998;BolovanFritts & Wiedeman, 2001), where virus infection does not spread to contiguous cells, resulting in a nonlytic persistent infection (Fish et al, 1998).…”

Morbidity in immunosuppressed (SCID/NOD) mice treated with reovirus (Dearing 3) as an anti-cancer biotherapeutic

“…30 Interestingly, human endothelial infection with cytomegalovirus may also be dependent on the vascular bed origin of the endothelium, 31 however, this finding has recently been disputed. 32 Furthermore, other mechanisms of viral oncolysis appear to use similar pathways as those exploited by reovirus. 33 One might expect then, that the use of SCID/NOD mouse models with these viruses may yield a similar "Black Foot" phenotype.…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…At the beginning of this study, the following findings were critical in directing interest to the ULb¢ region as a prime candidate: (i) the loss of EC-and leuko-tropism in the laboratory strains, Towne and AD169, was associated with the loss of the great majority of ULb¢ [52,53]; (ii) the low-passaged strain Toledo, which was shown to be tropism-deficient [51], was found to display an inversion of ULb¢ [53]; and (iii) extensive propagation of EC-and leuko-tropic clinical isolates in HELFs was found to be consistently associated with the selection of tropism-deficient variants [37,45,[54][55][56] in the presence of only minor variations in the relevant viral genomes.…”