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 oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.

G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300 000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein–Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.

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Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3 0 end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3 0 UTR of the (1) strand RNA. In contrast the 341 nucleotides at the 3 0 end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro.At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3 0 end of the minus-strand RNA: (a) the presence of a C residue as the 3 0 terminal nucleotide; (b) one or two G residues at positions 12 and 13; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3 0 end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.Keywords: HCV; RdRp; viral RNA; replication.Hepatitis C virus (HCV) is the major causative agent of transfusion-associated and sporadic non-A, non-B hepatitis [1]. More than 70% of HCV-infected patients develop chronic infection, often causing liver diseases such as chronic hepatitis, cirrhosis and hepatocellular carcinomas [2,3]. To date, the most effective treatment is a combination of interferon-a and the nucleoside analog ribavirin. However, only 40% of treated patients display a sustained biochemical response and inhibition of viral replication. Therefore, a more effective antiviral therapy is urgently required. HCV is a member of the family Flaviviridae. It is an enveloped virus with a single-stranded positive-sense RNA genome that contains a single long ORF translated as a polyprotein of about 3010 amino acids [4]. The ORF is flanked by two untranslated regions (UTR). The 341-nucleotide 5 0 UTR, together with the first nucleotides coding for the capsid, form an internal ribosome entry site (IRES) which is important for translation of the ORF. The 3 0 UTR is composed of a short variable region, a polyuridine tract of variable length and a 98-nucleotide sequence (3 0 X) which is highly conserved among various isolates [5]. It has been shown that this region is necessary for viral infectivity [6] but its exact role in viral replication is unknown.Studies of HCV replication have been hampered by the lack of efficient culture systems and by the fact that the only animal model is the chimpanzee [7,8]. More recently, a system allowing replication of a subgenomic fragment of hepatitis C virus RNA in a hepatoma cell line has been described [9]. Thus, most of the studies on the structures and functions of viral prote...

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The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.