In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

Abstract

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

Why large icosahedral viruses need scaffolding proteins

“…Discrete Model. The growth of the shells is based on the following assumptions (6,9,19,24): At each step of growth, a new trimer is added to the location in the boundary which makes the maximum number of bonds with the neighboring subunits. This is consistent with the fact that proteinprotein attractive interaction is weak and a subunit can associate and dissociate until it sits in a position that forms a few bounds with neighboring proteins.…”

“…A possible mechanism to successfully self-assemble a desirable structure might consist of protein subunits with chemical specificity, very much like in DNA origami (23) where structures with complex symmetries are routinely assembled. In viruses, however, capsids are built either from one or from a few different types of proteins, so specificity cannot be the driving mechanism leading to IO (9,18,21,24,25). In this paper, we show that a "generic" template provides a robust path to self-assembly of large shells with IO.…”

“…Under many circumstances, small icosahedral capsids assemble spontaneously around their genetic material, often a singlestranded viral RNA (5)(6)(7)(8)(9). However, larger double-stranded (ds) RNA or DNA viruses require what we generically denote as the template: scaffolding proteins (SPs) or an inner core (10)(11)(12)(13)(14)(15).…”

How a Virus Circumvents Energy Barriers to Form Symmetric Shells

“…The process of formation of virus particles in which the protein subunits encapsidate genome (RNA or DNA) to form a stable, protective shell called the capsid is an essential step in the viral life cycle [1][2][3]. The capsid proteins of many small single-stranded (ss)RNA viruses spontaneously package their wild-type (wt) and other negatively charged polyelectrolytes, a process basically driven by the electrostatic interaction between positively charged protein subunits and negatively charged cargo [4][5][6][7][8][9].…”

Cytoplasmic CPSF6 regulates HIV-1 capsid trafficking and infection in a cyclophilin A-dependent manner

“…Briefly, CA and CA-NC regions were amplified and subcloned into pET21 (EMD Chemicals Inc.) using NdeI and XhoI sites. Proteins were expressed and purified as previously described for Gag (ΔMA 15-100 Δ p6) (Ning et al, 2016;Zhao et al, 2013). CypA-DsRed-Exp2 (gift from Greg Melikyan) was cloned into the pET28 vector, resulting in a N-terminal His 6 -tagged protein.…”