Tracking hantavirus nucleocapsid protein using intracellular antibodies

Background 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. Methods 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. Result 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. Conclusion 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.


Background
Hantaviruses are members of the Bunyaviridae family, which contain three negative-sense, single-stranded RNA genome segments designated large (L), medium (M), and small (S) [1]. The S, M, and L segments encode the nucleocapsid protein (N), glycoproteins (Gn and Gc), and L protein (an RNA-dependent RNA polymerase), respectively. Hantaviruses do not have matrix proteins, but the N protein has been proposed to play a key role in virus assembly [2]. N protein is expressed in the cytoplasm, viral glycoproteins are co-translated in the endoplasmic reticulum (ER), once cleaved, Gn and Gc undergo glycosylation, folding, and heterodimerization in the Golgi complex, where they are retained and accumulate. For assembly to occur, N as well as Gn and Gc, must move to the same intracellular location. After interaction of N protein with viral RNA and subsequent assembly, ribonucleoprotein (RNP) is targeted to the Golgi complex by specific recognition of the cytoplasmic tail of Gn and Gc protein [3], the interaction of Gn protein cytoplasmic tail and the middle domain of the N protein was suggested to play essential role to direct RNPs to the site of the virus assembly [4] and the complete hetero-oligomeric (Gn-Gc) spike complex of hantaviruses might mediates the packaging of RNP into virions [5].
N protein has an intrinsic RNA chaperone activity, which is important for encapsidation and genome replication [6,7]. The RNA-binding domain of N protein is situated within a central conserved region between residues 175 and 217 [8]. The 141 residues proximal to the C-terminal are required for Golgi localization [9]. Both N-and C-terminal regions have been implicated in homotypic N protein interaction, and putative coiledcoil motifs in the N-terminal region of N protein have been proposed to facilitate trimerization [10][11][12]. N was not observed in the Golgi so far, but it could be observed to surround the Golgi after infection [13,9] and it was shown that targeting of N protein to the ER/ Golgi intermediate compartment (ERGIC), prior to its movement to the Golgi compartment, and an intact ERGIC are necessary for viral replication [14]. However, the impact of N protein intracellular trafficking on the cell and its effect on virus replication remain unclear. We used intracellular expression of anti-Hantaan virus (HTNV) and Seoul virus (SEOV) N protein N-terminaland C-terminal-specific antibodies, respectively, to block or knock down N protein function at targeted sites, with or without co-expressed membrane glycoproteins, and assess the effect on virus replication and N protein intracellular trafficking. Our data showed that N protein co-localized with both cytoplasm and ER-retarded antibodies either with or without the help of G protein and virus replication was inhibited by related intracellular antibodies. These data suggest, therefore, that presentation of N protein both in the cytoplasm and within the ER/cis-Golgi plays an important role in hantavirus replication.
Plasmid pEF-N76118 was generated by amplifying the HTNV strain 76-118 N protein coding sequence using primers carrying adapters, and then cloned into pEF/ myc/cyto and digestion with NcoI and XbaI.

Enzyme-linked immunosorbent assay (ELISA)
To determine the concentration of viral particles in culture supernatants, group-specific L13F3 mouse anti-N mAb was coated onto 96-well microtiter plates overnight at 4°C. Coated plates were blocked with 5% (w/v) skimmed milk in PBS, and serial dilutions of detergenttreated culture supernatants were added. After washing, bound viral particles were detected by horseradish peroxidase-conjugated L13F3 mAb diluted 1:1000 in PBS-T. After washing three times with PBS-T, TMB peroxidase substrate [100 μl; dimethylformamide and hydrogen peroxide (H 2 O 2 )] was added and developed at RT for~30 min. The reaction was stopped by adding H 2 SO 4 (100 μl; 1 N). A 450 (reference 620 nm) was read using a DTX 880 multi-mode detector (Beckman Coulter, Fullerton, CA, USA). The cutoff value was three standard deviations above the mean absorbance of the negative control wells.

Virus infection
HTNV strain 76-118 and SEOV strain L99 were propagated on Vero E6 cells. Cell cultures were harvested at 10 days postinfection; cell debris was removed by centrifugation at 4000 × g and samples stored in 1-ml aliquots at -80°C. Vero E6 cells and the intracellular antibodyexpressing Vero-E6 cell lines L13F3-scFv-ER-E6, H34-scFv-ER-E6, L13F3-scFv-CYTO-E6, and H34-scFv-CYTO-E6 were cultured in T25 flasks to 85% confluence; cells were washed twice with pre-warmed serumfree DMEM and virus 76-118 and L99 were loaded at a multiplicity of infection (MOI) level of 0.01. Cells were then incubated at 37°C for 2 h. Cells were washed twice with pre-warmed serum-free DMEM and maintained in DMEM (5 ml) supplemented with 2% (v/v) FBS and incubated at 37°C for 8 days. Cell culture supernatants (1.0 ml) were collected each day after the third day postinfection and an identical amount of fresh medium was added. Viral antigen levels were assayed by ELISA using the L13F3 mAb, as described above. Supernatant Hantaviruses titers were determined by serially diluting supernatants of each virus stock and performing quadruplicate infections on Vero-E6 cells in 96-well plates. The TCID50 assay endpoint was determined on day 10. Used medium was discarded and cells fixed in 80% (v/v) ice-cold acetone. Viral antigen were detected by horseradish peroxidase conjugated L13F3 monoclonal antibody, TMB peroxidase substrate (Dimethylformamide and hydrogen peroxide) was added to develop color. Absorbance at 450 nm (reference 620 nm) was read with a DTX 880 multi-mode detector (Beckman Coulter). The cut-off value was determined by the 3 times SD above the mean absorbance of the negative control wells. And these were addressed at the part of methods. The TCID50 value was calculated by the method of Reed and Muench [18].

Immunofluorescence assay (IFA)
Specificity of the scFvs and intracellular distributions of N protein and intracellular scFvs were determined by indirect immunofluorescence assay (IFA). Hantavirusesinfected Vero E6 cells, stably transfected Vero E6 cells, or transiently transfected COS-7 cells were passaged onto sterile coverslips in six-well plates overnight. Cells were fixed by treatment with methanol for 10 min at -20°C followed by acetone for 10 min at -20°C. To eliminate nonspecific binding, cells were pre-incubated with PBS containing 5% (v/v) FCS for 30 min at RT in a humidity chamber. Antibody dilutions of 1:50 for H34 IgG, 1:100 for L13F3 mAb, 5 μg/ml of purified anti-N protein scFvs, and 1:200 for anti-penta-His mAb in PBS containing 5% FCS were used. The first round of incubation was carried out for 1 h at RT. For detection of bound, purified anti-N protein scFvs, the secondary antibody (mouse anti-His tag mAb) was loaded and incubated for 1 h. Antibodies were detected using either an antihuman fluorescein isothiocyanate (FITC)-conjugated secondary antibody or anti-mouse tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antibody. Coverslips were washed in PBS, mounted on glass slides, and imaged under a TCS NT confocal microscope (Leica, Wetzlar, Germany).

Intracellular expression of L13F3, H34 scFvs
To determine the binding activity of the recombinant scFvs, immunoblot and IFA and ELISA analyses on HTNV strain 76-118 and SEOV strain L99 were performed. Figure 1 depicts an immunoblot using L13F3 ( Figure 1A, left panel) and H34 scFvs ( Figure 1A, right panel) to probe viruses. The L13F3 scFv showed binding to both strain 76-118 and L99; this finding was corroborated by IFA data ( Figure 1B, panel 1 and 2). The H34 scFvs bound only to HTNV strain 76-118 ( Figure 1B,  panel 3) and not SEOV stain L99 ( Figure 1B, panel 4) in the IFA test, and no binding was detected by immunoblotting ( Figure 1A, right panel). These data are consistent with the binding activities of the L13F3 and H34 parent antibodies.
Intracellular localization of scFvs was determined by indirect immunofluorescence. As expected, L13F3 and H34 scFvs-Cyto accumulated in the cytoplasm of transfected cells ( Figure 1C, 1 and 2), while L13F3 and H34 scFvs-ER yield a typical ER specific morphological fluorescence image ( Figure 1C, 3 and 4).

N protein interacted with intracellular antibodies in vivo
To investigate whether N protein interacts with intracellular scFvs in vivo, a His-tag pull-down experiment was performed. To exclude any nonspecific binding to Hisbind resin, 76-118 or L99 virus-infected Vero-E6 cells were used as negative controls and normal Vero-E6 cells as blank controls. N protein and intracellular scFvs were detected by SDS-PAGE analysis of the pull-down eluates (Figure 2A, lanes 2, 4, and 6). However, scFv H34, which proved to be a HTNV type-specific antibody, did not pull-down the N protein of Seoul L99 virus, as expected ( Figure 2A, lane 8). N protein and scFvs were not found in either negative or blank controls (Figure 2A, lanes 3 and 5). These observations were confirmed by detection with either anti-N protein mAb ( Figure 2B) or anti-His mAb ( Figure 2C) in immunoblot assays. Therefore, N protein and the N proteinspecific scFvs likely specifically interact in vivo.

Hantavirus N protein co-localized with intracellular antibodies
Intracellular localization of N protein in transiently cotransfected COS-7 cells or virus infected Vero-E6 cells containing intracellular antibodies was determined under a confocal microscope. The recombinant N protein of HTNV strain 76-118 co-localized with intracellular L13F3 and H34 scFvs in both the ER and cytoplasm in COS-7 cells ( Figure 3A). In contrast, the cytoplasmically expressed scFv did not co-localize with Gc protein of HTNV strain 76-118, but the ER-specific scFvs showed partial co-localization ( Figure 3B). The N protein of HTNV strain 76-118 also co-localized with scFvs in Vero-E6 cell lines with L13F3-ER, L13F3-CYTO, H34-ER, and H34-CYTO antibodies, and L99 viral N protein co-localized only with L13F3 derived intracellular antibodies ( Figure 3C).

Inhibition of N protein trafficking reduced HTNV replication
To determine the effect of intracellular antibodies on hantavirus replication, stably transfected L13F3-scFv-ER-E6, H34-scFv-ER-E6, L13F3-scFv-Cyto-E6, and H34-scFv-Cyto-E6 cell lines (1 × 10 6 ) were infected with HTNV strain 76-118 and SEOV strain L99 at a MOI of 0.01. Cell culture supernatants were collected each day after the third day postinfection and viral antigen loads were determined by ELISA. Data suggested that HTNV N protein antigen levels in L13F3-scFv-ER-E6 and L13F3-scFv-Cyto-E6 cell lines were lower than that of control cells ( Figure 4A). In contrast, N protein antigen levels in stably transfected H34-scFv-ER-E6 and H34-scFv-Cyto-E6 cell lines decreased only when infected with HTNV strain 76-118, but not with SEOV strain L99 ( Figure 4B). These data correlate with the antigen-binding specificities of the antibodies. No significant difference of virus replication was observed between stably transfected cell lines and normal non-transfected Vero E6 cells when intracellular antibodies were not specific for the infecting virus. This indicates that intracellular antibodies did not have a negative effect on cell growth, since the SEOV replication rate in H34 antibody-expressing cell lines was similar to that in control Vero E6 cells. HTNV levels in supernatants from the sixth day postinfection were titrated via TCID50 assay. Titers correlated with N protein antigen levels as determined by ELISA ( Figure 5). These data indicate that both ER-and cytoplasm-targeted scFv antibodies inhibited HTNV replication. However, HTNV type-specific H34 antibody did not significantly affect replication of SEOV strain L99.

Discussion
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 coiledcoil 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 ERtargeted 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  Titration of viral load in cell culture supernatants by TCID50 assay. Intracellular antibody-expressing stable cell lines were infected with hantavirus 76-118 and L99 respectively; virus titer at sixth day post infection was determined by the TCID50 assay. Endpoint was determined on day 10, cells fixed on the bottom of the culture plate by 80% (v/v) ice-cold acetone. Viral antigen were detected by horseradish peroxidase conjugated L13F3 monoclonal antibody, virus infection was assessed by absorbance at 450 nm (reference 620 nm). Error bars represent the standard deviation of three independent experiments. 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.