1074 are responsible for the occurrence of HFRS and HPS. Although Hantaviruses have been known to infect humans for almost a century, no specific therapeutics are available for the treatment of HFRS and HPS. Recent studies suggest that cell type-specific internalizing antibodies delivery of therapeutic genes is potentially a powerful strategy, given that a direct attack of the infected cells results in the blockade of in vivo viral propagation and even the elimination of the virus. In particular, chimeric proteins, which combine the nucleic acid-binding properties of the small basic protein, protamine, with the specific ligand- recognition properties of antibodies, have been engineered to deliver nucleic acid drugs to HIV-infected cells, HER2 over-expressing cancers (Song et al. 2005), activated LFA- 1-expressing cells (Peer et al. 2007), and HBV-infected cells (Wen et al. 2007a, b) in cell culture and animal models. Antibody fragments fused to lipids also have been used for effective retargeting of liposome-encapsulated DNAs in cancer therapy (Pirollo et al. 2006, 2007; Brignole et al. 2004). Taken together, these results indicate the potential utility of antibody-targeted system for intracellular trafficking of innovative therapeutic agents. Hantavirus infection is mediated by the interaction of viral surface GPs with integrin αvβ3 expressed on the host cell surface and subsequent endocytosis (Cheresh 1987; Hynes 1992). Hantavirus GPs include two transmembrane proteins, G1 and G2, which result from posttranslational cleavage of a single glycoprotein precursor and form heterodimers in the endoplasmic reticulum during the intracellular assembly of virus particles (Antic et al. 1992). Both G1 and G2 are normally located on the surface and in the cytoplasm of virus-infected cells (Ogino et al. 2004). Featured by the surface expression of these GPs, Hantavirus-infected cells could be targeted by therapeutics containing the corresponding antibodies. Engineered antibody fragments such as Fabs and scFvs, which have a lower molecular weight while possessing the affinity to the antigen, have been widely used in targeted therapy and drugs delivery. The prokaryotic E. coli expression system, characterized by easy genetic manipu- lation and scale-up, and relatively short duration between transformation and protein purification, is among the most popular host for recombinant protein production (Arbabi- Ghahroudi et al. 2005). In the cytoplasm of E. coli, it is preferable to express antibody fragments in soluble and active form, which can be achieved by one of the following fusion protein strategies: (1) fusion to a polypeptide that is highly soluble and (2) fusion to an enzyme that catalyzes disulfide bond formation. The soluble fraction of fusion protein allows disulfide bond formation and proper folding of the native structure in the cytoplasm and increases the possibility of obtaining soluble proteins with preserved biological activity. Fusion thioredoxin to target protein has Appl Microbiol Biotechnol (2010) 86:1067–1075 been widely used to avoid inclusion body formation and obtain active proteins (LaVallie and McCoy 1995; Wen et al. 2007a, b; Moura-da-Silva et al. 1999; Wu et al. 2003). In this study, the scFv3G1 against HTNV GP was expressed by E. coli as a soluble fusion protein and was purified using immobilized metal affinity chromatograph. The purified scFv was proven active and showed specific binding to HTNV GP antigen and then could be internalized into HTNV-infected cells. Application of targeted and intracellular delivery of effector moieties requires antibodies capable of internalization, i.e., a receptor-mediated endocytosis via a variety of clathrin- dependent and clathrin-independent endocytic pathways. Our results revealed that the recombinant HTNV GP antibody, scFv3G1, can be internalized following antigen engagement primarily through a clathrin-dependent endocytic pathway similar to that observed with transferrin. Similar clathrin- dependent internalization has been reported for the scFv against HBV antigen (Wen et al. 2007a, b). In the clathrin- dependent endocytotic pathway, internalized molecules are delivered to the early endosome and then trafficked to the late endosome–lysosome pathway (Mellman 1996; Sorkin and Von Zastrow 2002). Therefore, the internalization character- istics of the scFv3G1 were sufficient for the intracellular delivery of therapeutic agents, which thus may be released to the endolysosomal compartment, followed by rapid perme- ation into the cytosol and nucleus. The successful generation and purification of the Hantavirus glycoprotein antibody, scFv3G1, together with the demonstration that it can be internalized into HTNV-infected cells, suggests potential application of this recombinant antibody in targeted and intracellular delivery of effector moieties against HTNV infection. Acknowledgments This work was supported by grants from the National Basic Research Program of China (2004CB518805) and the National Natural Science Foundation of China (30872400). We are grateful to Yunqing Li and Le Niu for confocal microscopy and Mengyuan Shi, Haitao Wang, Weihong Wen, and Tao Wang for technical help. 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