Effect of C- and N-Terminal Polyhistidine Tags on Aggregation of Influenza A Virus Nuclear Export Protein

---

Olga N. Koroleva¹, Natalya V. Kuzmina², Anna P. Tolstova³, Evgeniy V. Dubrovin^4,5,a*, Valerii L. Drutsa⁶

¹Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

²Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 119071 Moscow, Russia

³Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

⁴Faculty of Physics, Lomonosov Moscow State University, 119991 Moscow, Russia

⁵National University of Science and Technology MISIS, 119049 Moscow, Russia

⁶Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received: January 25, 2024; Revised: November 6, 2024; Accepted: November 20, 2024

---

Nuclear export protein (NEP) of the influenza A virus, being one of the key components of the virus life cycle, is a promising model for studying characteristics of formation of amyloids by viral proteins. Using atomic force microscopy, comparative study of aggregation properties of the recombinant NEP variants, including the protein of natural structure, as well as modified variants with N- and C-terminal affinity His₆-tags, was carried out. All protein variants under physiological conditions are capable of forming aggregates of various morphologies: micelle-like nanoparticles, flexible protofibrils, rigid amyloid fibrils, etc. His₆-tag attached to the C-terminus has the greatest effect on aggregation kinetics and morphology of nanoparticles, which indicates important role of the C-terminal domain in the process of protein self-assembly. Molecular dynamics simulation did not reveal substantial influence of the His₆-containing fragments on the protein structure, but demonstrated some variations in the mobility of these fragments that may explain the observed differences in the aggregation kinetics of the different NEP variants. Hypothetical mechanisms for formation and interconversion of various aggregates are considered.

---

KEY WORDS: influenza A virus, nuclear export protein NEP, protein aggregation, poly(His)-tags, atomic force microscopy

DOI: 10.1134/S0006297924120125

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

---