FIG. 1.

Expression and self-assembly of NLV capsid proteins. HV and SMV genomic RNA was isolated from the stools of SMV- or HV-infected human volunteers. The capsid genes were isolated by reverse transcription-PCR and were subcloned into the pVR21 VEE replicon vector as previously described for NV1 (3) with specific primer pairs 5′-AGTCTAGTCCGCCAAGATGAAGATGGCGTCGAATGAC-3′ and 5′-NNNTTAATTAATTATTGCACTCTTCTGCGCC-3′ (HV-5′ and HV-3′, respectively) for HV or HV-5′ and 5′-NNNNNNNGGCGCGCCTTACTGAACCCTTCTACGC-3′ (SMV-3′) for SMV. (A) Amino acid (aa) alignment of NV, HV, and SMV ORF2 regions. NV is a genogroup I isolate, whereas SMV and HV are genogroup II NLVs from distinct genogroup clusters (GII.2 and GII.1, respectively). Percentage of amino acid identities are shown, and arrows indicate amino acid variations from published sequences (12-14). The NV ORF2 capsid clone NV1 has been previously described and is identical to the published NV ORF2 amino acid sequence (9, 12). (B) BHK cells were infected with packaged VRPs encoding NV, SMV, or HV capsid proteins. Expression of NLV capsid proteins was determined by IFA with human antiserum directed to either NV, SMV, or HV. After determination of the VRP titers by IFA as described previously (3, 9), BHK cells were infected with VRPs encoding either NV, SMV, or HV capsid proteins at a multiplicity of infection of 2. At 36 h postinfection BHK cells were lysed by freeze-thaw and the extracts were purified through sucrose gradients and were analyzed by negative-stain electron microscopic analysis (C). Scale bar, 100 nm.