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.

We used the polymerase chain reaction (PCR) to study which step(s) of the human immunodeficiency virus type 1 (HIV‐1) life cycle may be blocked following treatment of HIV‐exposed CEM cells with 13B8‐2, a monoclonal antibody (mAb) specific for the immunoglobulin (Ig) CDR3‐like region of the CD4 molecule and able to inhibit the productive infection of CEM cells by HIV‐1. The presence of viral RNA was investigated and found in 13B8‐2 mAb‐treated CEM cells 30 min after viral exposure; the full‐length viral DNA was found at 24 h post‐infection. We also found integrated forms of viral DNA at 24 h post‐infection. However, the integrated provirus was transcriptionally inactive in 13B8‐2 mAb‐treated cells, as demonstrated by the absence of spliced HIV‐1 mRNA. The lack of HIV transcription under 13B8‐2 mAb treatment was confirmed by chloramphenicol acetyltransferase (CAT) assay. We conclude that the inhibition of viral gene transcription accounts for the lack of progeny virions in culture supernatants of cells treated with this anti‐CD4 mAb. We also demonstrate that 13B8‐2 blocks viral production from chronically infected cells and restores CD4 cell‐surface expression on CEM cells containing an integrated provirus(es). We found this effect to be reversible. Moreover, we demonstrate that 13B8‐2 mAb treatment is efficient on different HIV‐1 and HIV‐2 virus isolates. These results may have major implications for the treatment of AIDS.

Nucleocapsid (NC) protein NCplO of Moloney murine leukemia virus is encoded by the 3' domain ofgag and contains a zinc finger surrounded by basic amino acids. During virion assembly, NC protein is necessary for core formation and the NC zinc finger is required for the packaging of the genomic RNA dimer. In vitro NCplO has RNA-binding and-annealing activities critical for virus infectivity, since NCplO promotes dimerization of viral RNA containing the Psi packaging element and annealing of replication primer tRNA"" to the initiation site of reverse transcription (primer-binding site). To investigate the role of the basic amino acids flanking the NCplO zinc finger, neutral residues were substituted for the basic amino acids and the effects of these mutations in vivo on virus assembly and infectivity and in vitro on the RNA-annealing activity of NCplO were analyzed. Here we report that the substitution of 1 or 2 neutral amino acids for the basic residues did not impair the production of mature virions but that infectivity was either moderately or strongly attenuated. When more than 2 basic residues were replaced by neutral amino acids, viruses were poorly infectious because of a severe defect in genomic RNA dimer packaging and initiation of reverse transcription. In vitro NCp1O-derived peptides with similar mutations were chemically synthesized and were found to be either fully or partially active or completely inactive. These data indicate that the basic residues flanking the zinc finger of NCplO are required for the production of infectious Moloney murine leukemia virus virions.