Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 U(L)31 and U(L)34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the U(L)34 protein are sufficient to mediate an interaction with the U(L)31 protein. A recombinant virus (v3480) lacking U(L)34 codons 138 to 181 was constructed. Similar to a U(L)34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A U(L)34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of U(L)31 protein in cells infected with a U(L)34 null virus, the U(L)31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the U(L)34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a U(L)31 null virus. In transient expression assays, the interaction between U(L)34 amino acids 137 to 181 and the U(L)31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the U(L)31 protein. These data indicate that amino acids 137 to 181 of the U(L)34 protein are (i) sufficient to mediate interactions with the U(L)31 protein in vitro and in vivo, (ii) necessary for the colocalization of U(L)31 and U(L)34 in infected cells, and (iii) essential for normal viral replication.