Concluding Remarks-Our data show that adenovirus specifically targets aspects of the nucleolar proteome in contrast to nonspecific disruption by agents like ActD, providing the first high throughput insight into viral-nucleolar interactions. Moreover, by examining the nucleolar proteome, we were able to highlight misdistribution of antigens from all compartments of the cell. We believe this demonstrates the utility of proteomics approaches to generate meaningful data sets that will help us understand the role of the nucleolus in viral infection and to uncover previously unsuspected effects on the host cell during viral infection. Indeed, subcellular fractionation of this sort coupled with confocal microscopy provides a powerful new tool for understanding viral host cell interactions in a broad context and is a positive step toward a systematic understanding of virus host cell interactions. Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design. Neutralizing antibodies provide one arm of the adaptive immune response against the human immunodeficiency virus type 1 (HIV-1). Several reports demonstrated that the neutralizing antibody response exerts selective pressure during HIV-1 replication in vivo, which accounts in part for the extensive variation in the env gene observed soon after primary infection [1, 2] . Furthermore, selective pressure imposed by neutralizing antibodies has been demonstrated in a human trial where three neutralizing monoclonal antibodies (mAbs) administered during HAART treatment-interruption led to a reduction in viremia followed by selection of escape mutants [3, 4] . Passive transfer studies in macaques showed that the administration of HIV-1 neutralizing mAbs protects against vaginal or intravenous challenge with SIV-HIV-1 chimeric viruses (SHIV) [5, 6, 7, 8, 9] . In some models protection depended not only on viral neutralization but also on Fc-mediated antibody effector functions [10, 11] .