Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview

Abstract

Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.

The CERV protein of Cer1, a C. elegans LTR retrotransposon, is required for nuclear export of viral genomic RNA and can form giant nuclear rods

“…That study showed Cer1 gRNA is highly abundant in gonad cytoplasm, but did not determine whether any of the cytoplasmic gRNA was associated with GAG particles [ 24 ]. Because retroviral capsids package only two molecules of single stranded gRNA [ 54 ], we re-examined gRNA localization using smFISH (single molecule fluorescence in situ hybridization) in combination with immunostaining for GAG. Mitotic nuclei typically showed 1–2 foci of gRNA, but staining in pachytene nuclei could usually be resolved into four, closely spaced foci which presumably represent each of the four LGIII chromatids ( Fig 2C , panels 1 and 2).…”

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity

“…Genome packaging in HIV-1 is dependent on interactions between an un-cleaved precursor PR55 NC domain and a 110 nucleotide Psi (ψ; RNA packaging signal) segment [ 94 ] in the 5′ RNA leader sequence [ 83 ]. The ψ segment comprises four stem loops (SL1–SL4) that are connected by short linkers [ 95 ]. The stem loops are proposed to have independent but redundant functions [ 94 ] which include the facilitation of genomic RNA dimerization by the dimer initiation site (DIS) in SL1; the packaging of un-spliced mRNAs when the signal is overlapped with the ψ-site by the major splice donor (SD) in SL2; heterologous RNA packaging by SL3; and the adoption of alternate structures by SL4 [ 22 ].…”

“…HIV-1 p6 comprises two unique late domains [ 102 ]. As part of the precursor PR55 Gag polyprotein, these domains recruit and bind the endosomal sorting complex required for transport (ESCRT) cellular factor to promote the abscission of immature virions from the host cell [ 95 ]. The first late domain, YPXL (Tyr-Pro-X-Leu), binds cellular factor ALG2-interacting protein X (ALIX) [ 105 ].…”