Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5′ end of the RNA genome. An RSV mutant with a Val‐Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and transacting factor, nucleocapsid protein, and thus might be linked during virion formation. Cells infected with Moloney murine leukemia virus (Mo-MuLV) produce three major size classes of virus-specific RNAs that are the 22S, 35S, and 70S RNAs (5). Most of the 22S and 35S RNA is found in free cytoplasm, while most of the 70S RNA is in the membrane-bound fraction (9). The MoMuLV 22S RNA is the messenger for the Pr85env precursor and is not found in virions. The 35S RNA is the genome-length RNA of 8,332 nucleotides (nt) which assumes two functions: it is the messenger RNA for the Pr659'9 and

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.

In addition to the Gag-Pol and Env precursors whose translation initiates at AUG codons, murine, feline, and simian type C oncoviruses also express glycosylated Gag-Pol precursors (glycoGag), glycoGag translation is initiated at CUG codons located upstream of the Gag AUG initiation codon. In contrast to Gag, glycoGag is translocated into the endoplasmic reticulum and is absent from virions. Since glycoGag has been described to be dispensable ex vivo, we investigated the in vivo effects of a glycoGag- mutation in the Friend murine leukemia virus (F-MuLV). F-MuLV induces severe early hemolytic anemia and subsequent erythroleukemia within 2 months after inoculation of newborn mice. We obtained a glycoGag- F-MuLV, strain H5, by inserting an octanucleotide linker downstream of the CUG codon leading to the reading of a stop codon in all reading frames upstream of the Gag AUG. F-MuLV H5 did not induce severe early hemolytic anemia, and latency of erythroleukemia was significantly increased most likely because of an approximately 1-week delay in the in vivo spreading. Accordingly, induction of recombinant polytropic viruses was also significantly delayed. Close examination of ex vivo spreading kinetics also showed a slower dissemination of F-MuLV H5. Western blot (immunoblot) performed after inoculation of newborn mice with this glycoGag- virus indicated the emergence of new glycoGag+ viruses. PCR analyses with F-MuLV-specific primers demonstrated in vivo pseudoreversions restoring the glycoGag reading frame. Our results demonstrated that glycoGag expression is positively selected and essential for full spreading and pathogenic abilities.