Role of annexins in cytomegalovirus infection I Annexin 2 tetramer enhances infection II Annexins 5 and 1 inhibit infection

Abstract

Cytomegalovirus (CMV), a widespread pathogen, causes significant pathology in immunocompromised individuals, and chronic infection has been linked to heart disease. Since CMV infection can be controlled, but not prevented, therapeutics aimed at the virus-cell interaction would be beneficial, but CMV-host cell interactions are not well understood. Cell-free experiments have suggested a function for annexin 2 (AnxA2), which exists in heterotetrameric (p36 2p112, AnxA2t) or monomeric (p36) form, but a role for AnxA2 in productive infection of cells is controversial. In the first part of this study we followed the effect on various stages of infection of endogenous AnxA2, using inhibitory antibodies, and purified AnxA2. Anti-p11 or anti-p36 antibodies inhibited CMV production by up to 95%,immediate-early (IE72) expression by 90% and plaque formation by 50%, but 35S-CMV-cell binding was unaffected. Confirming the role of endogenous AnxA2, AnxA2t and p11, but not p36, enhanced virus production 7-fold and IE72 expression 3-fold. Interestingly, purified proteins did not alter plaque formation, whereas AnxA2t, but not p36, enhanced 35S-CMV-cell binding 3-fold, suggesting biochemical differences between endogenous and purified AnxA2. IE72 expression was enhanced 3-fold in the p36-null cell line HepG2 following p36 transfection. Co-immunodepletion, chemical crosslinking, and ligand blots suggested an interaction between p11 and the viral fusogen glycoprotein B. In the second part of this study, we investigated whether purified AnxA5 and AnxA1, which interact with AnxA2, affect CMV infection by altering AnxA2 function. AnxA1 or AnxA5 inhibited CMV production by up to 80%, IE72 expression by 60% and plaque formation by 50%. However, 35S-CMV-cell binding was unaffected. Combined AnxA5 and AnxA1 were not additive at saturating concentrations, indicating that they inhibit by a similar pathway. Inhibition was reversed by preincubation with purified AnxA2t, and by anti-AnxA2 mAbs that alone had no effect on infection. Attenuation by AnxA1 or AnxA5 was not additive with inhibitory anti-AnxA2 antibodies, implying that a common pathway, i.e. AnxA2t, was impeded. AnxA1 or AnxA5 inhibited IE72 gene expression by 80% in p36-transfected but not native HepG2. These data demonstrate that (1) AnxA2t enhances CMV infection, and (2) purified AnxA5 and AnxA1 inhibit CMV infection by disrupting AnxA2t activity.