Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.

Sixty-six 20- to 23-amino-acid synthetic peptides, partially overlapping by 10-12 amino acids, spanning the entire sequence of the envelope SU and TM glycoproteins of the Petaluma isolate of FIV, have been used to investigate the Env domains involved in viral infection. Peptides 5 to 7, spanning amino acids 225E-P264 located in a conserved region of the SU protein, and peptides 58 to 61, spanning amino acids 767N-P806 and encompassing hypervariable region 8 of TM protein, exhibited a remarkable and specific antiviral effect against the homologous and one heterologous isolate, as judged by inhibition of FIV-induced syncytium formation and p25 production in CrFK cells. Peptides 5 and 7, but not peptides 58 and 59, also inhibited viral replication of a fresh FIV isolate on nontransformed lymphoid cells. By flow cytometry, peptides 5, 7, 58, and 59 were shown to bind the surface of FIV permissive cells. The antiviral activity of peptides 5 and 7, however, was time-dependent, as inhibition of FIV replication was seen when the peptides were administered before or within 3 hr after virus inoculation; in contrast, TM peptides 58 and 59 exerted a potent inhibitory effect when added up to 24 hr after virus inoculation. Circular dychroism analysis showed that peptide 5 folds to a helical conformation in the presence of a hydrophobic environment. Although the basis for the antiviral action of the peptides is not understood, our data suggest that the inhibitory peptides may act by interacting with cell-surface molecules involved in viral infection.

A battery of sixty-six 20- to 23-amino acid synthetic peptides, partially overlapping by 10-12 amino acids, spanning the entire sequence of the envelope (Env) glycoproteins of the Petaluma isolate of feline immunodeficiency virus (FIV-Pet), has been used to map Env linear B cell epitopes. By screening FIV-infected cat sera for anti-peptide reactivity, the existence of two immunodominat domains, namely the V3 region of the surface (SU) glycoprotein and the domain including the highly conserved sequence QNQFF of the transmembrane (TM) glycoprotein, was detected; antibody-binding sites were also mapped in the domain overlapping the cleavage site between SU and TM encompassing the V6 variable region. Moreover, at least two novel linear B epitopes, the former spanning residues 427M-H446 and the latter spanning residues 737N-N756 and likely representing a "type-specific" determinant, have been revealed. The battery of synthetic peptides was then used to immunize outbred Swiss mice in the attempt to reveal other potential sites of immunogenicity of the Env glycoproteins. Analysis of peptide-immunized mouse sera for anti-peptide reactivity revealed more numerous B cell epitopes, generally mapping in different peptides, as compared with those defined in the feline system. None of the mouse anti-peptide sera, however, proved neutralizing for FIV-Pet.

No abstract