Tracking hantavirus nucleocapsid protein using intracellular antibodies

Hantavirus nucleocapsid (N) protein is a multifunctional viral macromolecule involved in multiple stages of the viral replication cycle. The intracellular trafficking of N protein during virus assembly remains unclear.

We used N protein-specific intracellular expressed antibodies to track the localization and distribution of Hantaan virus and Seoul virus N protein. The N protein-specific antibody single-chain variable antibody fragments (scFvs), which bind an N-terminal linear epitope (L13F3) and C-terminal conformational domain (H34), were intracellularly expressed in the endoplasmic reticulum (ER) by fusion of the SEKDEL retention signal peptide at the carboxyl terminus, and in the cytoplasm (Cyto) by deletion of the ER membrane target signal peptide. Stable Vero-E6 cell lines expressing intracellular scFvs were either infected with hantavirus or transfected with an N protein expression plasmid; virus replication and N protein intracellular localization were determined.

N protein co-localized with scFvs in the ER and cytoplasm with or without viral membrane glycoproteins. Hantavirus replication was inhibited in both the scFvs-ER- and scFvs-Cyto-expressing stable cell lines.

N protein may be expressed in the ER retention signal peptide of KDEL circulating region (ER/cis-Golgi) without the assistance of G protein, and so expression of N protein in both the cytoplasm and within the ER/cis-Golgi plays an important role in virus replication.