The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1 MN virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.Eliciting broadly neutralizing antibodies (Abs) to human immunodeficiency virus (HIV-1) is a major goal of vaccine research but one that has proved extraordinarily elusive (8,11,77). This probably reflects the low antigenicity and immunogenicity of the HIV-1 envelope spike and most especially of relatively conserved regions of the spike. It is clear that much of the protein surface of the gp120 and gp41 protein molecules in the heterotrimeric envelope spike (gp120 3 -gp41 3 ) is directly or indirectly occluded from Ab binding by protein-protein interaction. Thus, for example, extensive surfaces on gp41 appear to be involved in interaction with other gp41 molecules and with gp120 (62, 63). Reciprocally, a portion of the surface of gp120 is occluded by the interaction with gp41 and by trimer formation (28,32,75,76). The relatively low immunogenicity of HIV-1 envelope trimers is also inferred from the low titers of neutralizing Abs, particularly cross-isolate neutralizing Abs, elicited during natural infection (31,39,40). This follows since a good correlation has been established between Ab neutralization and binding to envelope spikes, at least for T-cell-line-adapted viruses (50, 57, 61), suggesting that the deficit in neutralization originates from a deficit in spike binding. Low immunogenicity presumably arises, at least in part, from the weakly stimulating properties of the exposed regions of the envelope trimer. These include extensive regions of carbohydrate. A caveat here is that one cannot generally be sure of the eliciting antigen: Abs reactive with the trimer may have been elicited by other...

SummaryPoly(ADP-ribosyl)ation is an immediate DNA-damagedependent post-translational modification of histones and other nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a large family of 18 proteins, encoded by different genes and displaying a conserved catalytic domain in which PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are so far the sole enzymes whose catalytic activity has been shown to be immediately stimulated by DNA strand breaks. A large repertoire of sequences encoding novel PARPs now extends considerably the field of poly(ADP-ribosyl)ation reactions to various aspects of the cell biology including cell proliferation and cell death. Some of these new members interact with each other, share common partners and common subcellular localizations suggesting possible fine tuning in the regulation of this posttranslational modification of proteins. This review summarizes our present knowledge of this emerging superfamily, which might ultimately improve pharmacological strategies to enhance both antitumor efficacy and the treatment of a number of inflammatory and neurodegenerative disorders. A provisional nomenclature is proposed.

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3,is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

We have investigated whether nonneutralizing monoclonal antibodies (MAbs) to the gp120 subunit of the envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) can interfere with HIV-1 neutralization by another anti-gp120 MAb. We used neutralizing (b12) and nonneutralizing (205-42-15, 204-43-1, 205-46-9) MAbs to the epitope cluster overlapping the CD4-binding site (CD4BS) on gp120. All the MAbs, neutralizing or otherwise, cross-competed for binding to monomeric gp120, indicating the close topological proximity of their epitopes. However, the nonneutralizing CD4BS MAbs did not interfere with the neutralization activity of MAb b12. In contrast, in a binding assay using oligomeric Env expressed on the surface of Env-transfected cells, the nonneutralizing MAbs did partially compete with b12 for Env binding. The surface of Env-transfected cells contains two categories of binding site for CD4BS MAbs. One type of site is recognized by both b12 and nonneutralizing CD4BS MAbs; the other is recognized by only b12. Binding assays for Env-gp120 interactions based on the use of monomeric gp120 or Env-transfected cells do not predict the outcome of HIV-1 neutralization assays, and they should therefore be used only with caution when gauging the properties of anti-Env MAbs.Neutralization of human immunodeficiency virus type 1 (HIV-1) involves the binding of antibodies to the native, fusion-competent envelope glycoprotein (Env) complex on the surface of infectious virions (27,45,48,56). Most of the known neutralizing antibodies whose epitopes have been characterized and whose mechanisms of action have been explored work by inhibiting the interactions of the virus with its receptors, CD4 and the CCR5 or CXCR4 coreceptor (45,66,70,71). In the case of the 2G12 monoclonal antibody (MAb) to a glycan epitope on gp120, inhibition of coreceptor binding probably occurs indirectly (52,57,70,72). The inhibitory actions of MAbs can occur prior to attachment of the virus to the cell surface or subsequent to a semispecific absorption of the virus to ancillary receptors such as heparin sulfate proteoglycans (62,70). An exception is the 2F5 MAb to a conserved epitope in the C-terminal region of the gp41 ectodomain, which probably neutralizes HIV-1 infectivity by interfering with receptormediated conformational changes in the envelope glycoproteins subsequent to the virus-receptor interactions (70). By analogy, the 4E10 and Z13 antibodies to epitopes proximal to the 2F5 site are likely to have a similar mechanism of action (63,74,75).