A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.