The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.