Study shows molecular details of HIV-1 assembly and incorporation of viral envelope proteins

Assembly of HIV-1, which causes AIDS, takes place on the inner plasma membrane leaflet of infected cells, a process of geometric construction that creates hexamers from the trimers of the viral protein Gag, guided by the domain N-terminal matrix of Gag.

Yet some details of this virion assemblage have been missing for four decades. In a study published in the journal Proceedings of the National Academy of SciencesJamil Saad, Ph.D., and colleagues provide the first atomic view of the matrix lattice, showing molecular detail at 2.1 angstrom resolution, a step that advances understanding of key mechanisms of viral assembly and the incorporation of viral envelope proteins.

“Our findings may facilitate the development of novel therapeutic agents that inhibit HIV-1 assembly, envelope incorporation, and ultimately virus production,” said Saad, a professor of microbiology at the University of Alabama to Birmingham.

The Gag protein is modified after translation, in which a lipid-like myristate group is added to help Gag bind to the plasma membrane. How the myristoylated matrix domain, or myrMA, of Gag assembles into a network has eluded detection until now.

Low molecular resolution techniques -; such as cryo-electron diffraction and cryo-electron tomography -; suggested that the myrMA protein organizes into trimers, and that these trimers then undergo higher-order organization to form trimer hexamers. Saad’s study is consistent with a recent study, which suggested that the myrMA protein undergoes dramatic structural changes to allow the formation of distinct hexamer networks in immature and mature virus particles. Virus maturation is the last step in the virus replication cycle, as the capsid core forms inside the assembled virus, producing infectious particles.

The HIV-1 envelope protein, or Env, is a transmembrane protein delivered to the plasma membrane by the secretory pathway of the cell. Most of the Env protein extends beyond the membrane, but a tail crosses the membrane back inside the cell. Genetic and biochemical studies have suggested that the incorporation of the viral Env protein into viral particles also depends on the interaction between the myrMA domain and the cytoplasmic tail of Env. In 2017, Saad’s lab resolved the high-resolution structure of the cytoplasmic tail of Env, which was the last unknown protein structure of HIV-1.

Env is a key infectivity protein. When a mature HIV-1 virus approaches a target cell, Env attaches to proteins outside the uninfected cell, then the Env protein snaps in like a mousetrap to fuse the viral membrane to the membrane cellular.

In the structures described by colleagues from Saad and UAB, the myristic acid of myrMA plays a key role in stabilizing the lattice structure, so the ability to form myrMA crystals was important. They solved this elusive technical challenge by removing 20 amino acids from the end of the 132 amino acid myrMA. The formation of a Gag network on the plasma membrane is known to be mandatory for the assembly of immature HIV-1 and Env incorporation.

Saad and co-workers report that their myrMA array is arranged as a hexamer of trimers with a central hole, believed to accommodate the C-terminal tail of Env to promote incorporation into virions. Their myrMA crystals allowed them to observe the attached myr group in the lattice. They found that the myr group of a myrMA subunit inserts into the subunit’s hydrophobic cavity through the dual axis, introducing “myristoyl exchange”, and they also reported other interactions molecules between the trimers. The researchers described additional molecular details that help stabilize the hexamer of the trimer network.

By conducting mutagenesis studies coupled with a nuclear magnetic resonance, or NMR, technique, the researchers demonstrated that a single amino acid substitution in the matrix -; Leucine-13 or Leucine-31 to a glutamic acid -; induces a conformational change in myrMA that can destabilize trimer-trimer interactions within the network. Previous genetic studies have indicated that leucine-13 or leucine-31 substitution has adverse effects on Env incorporation.

Another important finding in this study is evidence for an alternate membrane binding mechanism of Gag, which is known to be mediated by interactions of the myrMA domain with phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P.2, a lipid exclusively located on the inner leaflet of the plasma membrane. UAB researchers showed that PI(4,5)P2 is able to bind to alternative sites on MA. This is consistent with a novel mechanism of alternating MA-membrane binding to PI(4,5)P2 during assembly of the immature particle and during maturation.

“In conclusion, we provided an atomic view of the HIV-1 myrMA network that revealed invaluable structural information on myrMA subunit arrangement, trimers, trimer-trimer interface, myr exchange, the impact of Env incorporation defective MA mutations on myrMA structure and hence network formation,” Saad said. “Our data also supported an MA–PI(4,5)P alternation2 binding mechanism during virus assembly and maturation. These findings have filled a major gap in our understanding of the mechanisms of Gag assembly on the plasma membrane and Env incorporation into viral particles.”

Source:

University of Alabama at Birmingham

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