||After budding from infected cells, Human Immunodeficiency Virus type 1 (HIV-1) undergoes a maturation process that is required for viral infectivity. In the immature state, the major structural protein of viral particles, Gag, forms a thick protein shell underneath the viral membrane. During maturation, Gag is cleaved by HIV-1 protease and the resulting mature particles have a much thinner membrane-bound protein layer surrounding a conical core (capsid), which is a dramatic morphological change from the immature state. Employing atomic force microscopy (AFM), Itay Rousso's group together with ours previously discovered a dramatic change in particle stiffness during HIV-1 maturation mediated by the cytoplasmic tail (CT) of HIV-1's envelope protein (Env). A correlation between high particle stiffness and weak viral entry activity led us to investigate whether viral particle stiffness directly regulates viral entry activity. First, after observing that standard viral purification conditions perturb virion structure and stiffness, we determined new purification conditions that preserve both. Next, we showed that CT alone is sufficient to regulate viral particle stiffness. This observation allowed us to independently increase particle stiffness using a construct that lacks the Env ectodomain and thus has no entry ability itself. Using this system, we showed that particle stiffness directly regulates the entry activity of the Env proteins of both HIV-1 and the unrelated Vesicular Stomatatis Virus (VSV). These results suggest a general role for particle stiffness in the regulation of viral entry, linking viral physical properties and biological functions. Mutagenesis studies reveal the important domains within CT or Gag that regulate viral entry activity. Employing an Env mutant with cleavable CT, we obtained preliminary data about the timing of CT's regulation of particle stiffness. Taken together, these studies improve our understanding of viral structure and function during the viral lifecycle and suggest potential novel inhibitory strategies.