Assembly, part of the late stages of the retroviral life cycle, begins when the structural polyprotein Gag associates with viral genomic RNA. Ultimately, more than a thousand Gag molecules form a spherical immature virion. Maturation takes place soon after or concomitantly with virus budding and is initiated as Gag is cleaved by the retroviral protease into its constituent protein domains. The immature core is thought to disassemble and the liberated CA proteins to reassemble into a morphologically distinct mature capsid. In vitro assembly with derivatives of Gag and CA has been used to study retroviruses for over two decades. In this review, we examine the discovery and development of three major model systems [human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Mason–Pfizer monkey virus (MPMV)] and discuss structural features and aspects of the retroviral assembly pathway that have been uncovered using in vitro assembly. We also put forward two major unresolved questions in the field and propose future avenues of research.

Keyword(s): CAcapsidGagHIV-1MPMVRSV

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