In this study, we demonstrated that N protein of Hantaan and Seoul virus localizes to the ER/cis-Golgi in the absence of membrane glycoproteins. Data further suggested that N protein co-localized with intracellular antibodies with the ER retention marker SEKDEL. In addition, analysis of antibody-expressing cell lines, infected with viruses, revealed that N protein trafficking to the ER/cis-Golgi is important for viral replication. Blocking of N protein trafficking at both the ER/cis-Golgi and in the cytosol leads to inhibition of replication.
The N protein is a 420-430 residue 50-kDa protein, the N-terminal 75 residues of which carry two coiled-coil motifs that facilitate trimerization and nucleocapsid protein trimers that are believed to be HTNV particle assembly intermediates [
10,
11]. The L13F3 scFv antibody used in this study is known to bind to the N terminal 30 residues of N protein, which comprises part of the first coiled-coil motif [
11]. Additionally, H34 scFv has been shown to bind to a conformational epitope.
In hantaviruses, N protein is the first viral protein to accumulate during infection [
13,
19,
20]. Viral RNA segments are complexed with N protein to form individual L, M, and S nucleocapsids [
1], which are then packaged into the virion at the bilayered envelope within which are embedded the two viral surface glycoproteins Gn and Gc [
21]. The specific interaction between N protein and glycoprotein is thought to trigger budding of virions into the Golgi cisternae and to initiate the virus assembly[
4,
5]. Although the laboratory evidences were presented recently, the precise mechanism of N protein entry into the ER/
cis-Golgi apparatus remains unclear. ER-targeted scFvs used for tracking N protein intracellular trafficking were combined with a SEKDEL sequence at the carboxyl terminus. In mammalian cells, SEKDEL receptors are localized primarily to the early Golgi complex at steady state [
22,
23], but shift in their localization upon binding of SEKDEL-bearing ligands [
24]. Therefore, we predicted that the ER-targeted scFvs would follow a route from the ER to the
cis-Golgi and retrograde transport back to the ER. The co-localization and the proved interaction of N and ER-targeted intracellular antibodies indicate that N protein may entry into the ER/
cis-Golgi apparatus. During virus infection, N protein or nucleocapsids also co-localized with ER-targeted antibodies. Virus replication was inhibited by the ER-targeted antibodies, which implies that N protein or nucleocapsids presented in membrane cisternae at the ER/
cis-Golgi apparatus. Previous studies have shown that N protein is membrane-associated [
9,
14], and that both targeting of N to ERGIC prior to its movement to the Golgi compartment and an intact ERGIC were required for viral replication [
14]. In membrane subcellular fractionation experiments, a small proportion of total N protein was detected in membrane-containing fractions [
14]. We hypothesize that N protein enters the ER or
cis-Golgi apparatus, but only with low efficiency. An interaction between ER-targeted scFvs and membrane-associated N protein may act as a "motor" to enhance the entry of N protein into ER/
cis-Golgi membrane vesicles. Like the function of the interaction of N protein and the cytoplasmic tail of Gn/Gc during virus replication [
4,
5]. The ER provides a lower-energy environment than the Golgi system [
25], which might enhance the entry of cytosol protein or nucleocapsids. The interaction between N and G protein is not the only prerequisite for the entry of N protein or cytosol nucleocapsids into membrane vesicles. Antibodies or other molecules might be alternative driver to direct the N protein to membrane vesicles. Immunoelectron microscopy examination of Uukuniemi virus, a bunyavirus, also demonstrated that the nucleocapsid is associated with membranes that show the characteristic distribution and tubulovesicular morphology of the pre-Golgi intermediate compartment, suggesting that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack [
26].
In summary, the data we present in this study suggest that N protein may present in the ER/cis-Golgi without the assistance of viral G protein, and that N protein trafficking at these sites plays an important role in HTNV replication.