Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the first human virus known to encode dihydrofolate reductase (DHFR), an enzyme required for nucleotide and methionine biosynthesis. We have studied the purified KSHV-DHFR enzyme in vitro and analyzed its expression in cultured B-cell lines derived from primary effusion lymphoma (PEL), an AIDS-associated malignancy. The amino acid sequence of KSHV-DHFR is most similar to human DHFR (hDHFR), but the viral enzyme contains an additional 23 amino acids at the carboxyl-terminus. The viral DHFR, overexpressed and purified from E. coli, was catalytically active in vitro. The K(m) of KSHV-DHFR for dihydrofolate (FH(2)) was 2.4 microM, which is significantly higher than the K(m) of recombinant hDHFR (rhDHFR) for FH(2) (390 nM). K(m) values for NADPH were similar for the two enzymes, about 1 microM. KSHV-DHFR was inhibited by folate antagonists such as methotrexate (K(i): 200 pM), aminopterin (K(i): 610 pM), pyrimethamine (K(i): 29 nM), trimethoprim (K(i): 2.3 microM), and piritrexim (K(i): 3.9 nM). In all cases, K(i) values for these folate antagonists were higher for KSHV-DHFR than for rhDHFR. The viral enzyme was expressed at levels two- to tenfold higher than hDHFR in PEL cell lines as an early lytic cycle gene. KSHV-DHFR mRNA and protein appeared from 6 to 24 h after chemical induction of the KSHV lytic cycle. Epitope-tagged KSHV-DHFR and rhDHFR both localized to the nucleus of transfected cells, while other KSHV nucleotide metabolism genes localized to the cytoplasm. DHFR activity was not essential for viral replication in cultured PEL cells. Since hDHFR was not detectable in peripheral blood mononuclear cells (PBMCs), KSHV-DHFR may function to provide increased DHFR activity in vivo in infected cells that have little or none of their own enzyme.