Palindromic sequence plays a critical role in human foamy virus dimerization

The retroviral RNA genome is dimeric, consisting of two identical strands o f RNA linked near their 5' ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimeriz ation process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McCl ure, Virology 229:251-258, 1997). To characterize these sites further, a se ries of mutants were designed and tested for their ability to dimerize in v itro. The primer binding site and a G tetrad in SI were dispensable for dim erization. However, a mutant that changed the 3' end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition o f the SII palindrome, consisting of 10 nucleotides, proved to be critical f or in vitro dimerization, since mutations within this sequence or replaceme nt of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an impo rtant role. A moderate extension to 12 nucleotides was tolerated, whereas a n extension to 16 nucleotides or more impaired dimerization. When nucleotid es flanking the palindrome were mutated in a random fashion, dimerization w as unaffected. Changing the SIII sequence also led to decreased dimer forma tion, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimeriza tion in vitro also abolished virus replication. In contrast, constructs con taining mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant rev ealed reversion to the wild type but with the insertion of an additional tw o nucleotides. Analysis of cell-free virions demonstrated that both replica tion-competent and replication-defective mutants packaged nucleic acid. Thu s, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.