Role of the UL45 protein in herpes simplex virus entry via low pH-dependent endocytosis and its relationship to the conformation and function of glycoprotein B

Abstract

Herpesviruses commandeer distinct cellular pathways to enter target cells. The mechanism by which herpes simplex virus (HSV) selects a pH-dependent, endocytic route or a pH-independent route remains to be elucidated. We investigated the role of the non-glycosylated viral envelope protein UL45 in HSV entry via endocytosis. UL45 plays a role in mediating cell-cell fusion and has been proposed to functionally interact with gB to regulate membrane fusion. Thus, we also probed the impact of UL45 on the structure and function of gB present in virions. A UL45 deletion virus successfully entered cells via low pH, endocytic pathway with wild type kinetics. In the absence or presence of UL45, the antigenic conformation of virion gB appeared unaltered. Antibodies to gB neutralized infection of the UL45-deletion virus and wild type virus to a similar extent, regardless of whether the target cells supported low pH endocytic or non-endocytic entry routes. Lastly, HSV virions were inactivated by low pH regardless of the presence of UL45. The results, together with previous studies, suggest that UL45 plays distinct roles in cell-cell fusion and virus-cell fusion during acid-dependent entry.

Fig. 1

(A) Entry of HSV-1 KOS UL45Δ into CHO-nectin-1 cells. HSV-1 KOS or KOS UL45Δ was added to CHO-nectin-1 cells at MOIs ranging from 0 to 2. Beta-galactosidase activity of cell lysates was quantified at 6 hr post-infection as an indication of viral entry. Entry of the two viruses was not statistically different (p = 0.40, Student’s t-test). (B) Effect of ammonium chloride on the cell entry of HSV-1 KOS UL45Δ. CHO-nectin-1 cells were treated with indicated concentration of ammonium chloride for 30 min. HSV-1 KOS or KOS UL45Δ (MOI of 1) was added to cells for 6 hr in the continued presence of agent. Entry was measured as the percent of beta-galactosidase activity of cell lysates relative to that obtained in the absence of lysosomotropic agent. Data are means of quadruplicate determinations with standard deviation. Entry was not statistically different in the presence of NH⁴Cl (p = 0.94, Student’s t-test).

Fig. 2

Low pH inactivation of HSV-1 UL45Δ. Samples of HSV-1 strain KOS or KOS UL45Δ were adjusted to a range of pHs as shown, incubated at 37°C for 10 min, and then neutralized to pH 7.6. Treated virions were incubated with (A) HaCaT or (B) Vero cells, and plaque formation was measured as an indication of virus entry and infection. The infectivity of samples that were treated with pH 7.5 was defined as 100%. Inactivation of HSV-1 UL45Δ was not statistically different from wild type (p = 0.34 for HaCat and p = 0.91 for Vero, Student’s t-test).