The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

• Keywords
• CAH *, IP6 *, M-PMV *, MLV *, RSV *, SP domain *, assembly *, hexamer *, immature *, polyanion *,
• MeSH
• Alpharetrovirus physiology   MeSH
• Betaretrovirus physiology   MeSH
• Cell Membrane chemistry   metabolism   MeSH
• Gammaretrovirus physiology   MeSH
• Gene Products, gag chemistry   metabolism   MeSH
• Host-Pathogen Interactions * MeSH
• Cells, Cultured MeSH
• Polyelectrolytes chemistry   metabolism   MeSH
• Retroviridae physiology   ultrastructure   MeSH
• Virus Assembly * MeSH
• Virion MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, gag MeSH
• polyanions MeSH Browser
• Polyelectrolytes MeSH

Despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. Persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. A combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. Initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. We provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses.

• Keywords
• Assay, Assembly, Cell-based, Entry, High-throughput screening, In vitro, Inhibitor, Method, Replication, Virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Virus Physiological Phenomena drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Host-Pathogen Interactions drug effects   MeSH
• Virus Internalization drug effects   MeSH
• Capsid drug effects   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical methods   MeSH
• Virus Replication drug effects   MeSH
• High-Throughput Screening Assays methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antiviral Agents MeSH
• Enzyme Inhibitors MeSH

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.

• Keywords
• capsid, cryoelectron tomography, maturation, murine leukemia virus, retrovirus,
• MeSH
• Cryoelectron Microscopy MeSH
• Gene Products, gag chemistry   genetics   ultrastructure   MeSH
• HEK293 Cells MeSH
• HIV-1 chemistry   genetics   ultrastructure   MeSH
• Capsid chemistry   ultrastructure   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mice MeSH
• Protein Domains MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Electron Microscope Tomography MeSH
• Virion chemistry   genetics   ultrastructure   MeSH
• Capsid Proteins chemistry   genetics   ultrastructure   MeSH
• Leukemia Virus, Murine chemistry   genetics   ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Comparative Study MeSH
• Names of Substances
• Gene Products, gag MeSH
• Capsid Proteins MeSH