Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.

• Keywords
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• Cell Line MeSH
• Cysteine genetics   MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation MeSH
• Proviruses genetics   MeSH
• Virus Assembly * MeSH
• Virion physiology   MeSH
• Capsid Proteins chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cysteine MeSH
• Capsid Proteins MeSH

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

• MeSH
• Alcoholism drug therapy   epidemiology   MeSH
• Molecular Targeted Therapy MeSH
• Disulfiram chemistry   pharmacology   therapeutic use   MeSH
• Adult MeSH
• Nuclear Proteins chemistry   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Copper chemistry   MeSH
• Mice MeSH
• Neoplasms drug therapy   metabolism   mortality   pathology   MeSH
• Alcohol Deterrents * pharmacology   therapeutic use   MeSH
• Drug Repositioning * MeSH
• Protein Aggregates MeSH
• Proteolysis drug effects   MeSH
• Antineoplastic Agents * pharmacology   therapeutic use   MeSH
• Heat-Shock Response drug effects   MeSH
• Protein Binding drug effects   MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Denmark epidemiology   MeSH
• Names of Substances
• Disulfiram MeSH
• Nuclear Proteins MeSH
• Copper MeSH
• NPLOC4 protein, human MeSH Browser
• Alcohol Deterrents * MeSH
• Protein Aggregates MeSH
• Antineoplastic Agents * MeSH

UNLABELLED: The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE: Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

• MeSH
• Cell Line MeSH
• Cryoelectron Microscopy MeSH
• Genome, Viral * MeSH
• Gene Products, gag MeSH
• Humans MeSH
• Mason-Pfizer monkey virus physiology   ultrastructure   MeSH
• Mutation MeSH
• Recombinant Proteins MeSH
• RNA, Viral metabolism   MeSH
• Amino Acid Sequence MeSH
• Virus Assembly * genetics   MeSH
• Amino Acid Substitution MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, gag MeSH
• Recombinant Proteins MeSH
• RNA, Viral MeSH
• Capsid Proteins MeSH