Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus

Marked interstrain differences in the endothelial cell (EC) tropism of huma n cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of HCMV that determines this p henotype. The infection efficiency of various HCMV strains in EC versus fib roblasts was quantified by immunodetection of immediate early (IE), early a nd late viral antigens. Adsorption and penetration were analysed by radiola belled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic t ranslocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of vira l IE antigens. The infectivity of non-endotheliotropic HCMV strains in EC w as found to be 100-1000-fold lower when compared to endotheliotropic strain s. The manifestation of this phenotype at the level of IE gene expression i ndicated the importance of initial replication events. Surprisingly, no int erstrain differences were detected during virus entry. However, dramatic in terstrain differences were found regarding the nuclear translocation of pen etrated viral DNA, With nonendotheliotropic strains, the content of viral D NA in the cell nucleus was 100-1000-fold lower in EC when compared to endot heliotropic strains, thereby reflecting the strain differences in IE gene e xpression. Simultaneous staining of viral particles and viral IE antigen re vealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.