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A requisite step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the insertion of the viral genome into that of the host cell, a process catalyzed by the 288-amino-acid (32-kDa) viral integrase (IN). IN recognizes and cleaves the ends of reverse-transcribed viral DNA and directs its insertion into the chromosomal DNA of the target cell. IN function, however, is not limited to integration, as the protein is required for other aspects of viral replication, including assembly, virion maturation, and reverse transcription. Previous studies demonstrated that IN is comprised of three domains: the N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD). Whereas the CCD is mainly responsible for providing the structural framework for catalysis, the roles of the other two domains remain enigmatic. This study aimed to elucidate the primary and subsidiary roles that the CTD has in protein function. To this end, we generated and tested a nested set of IN C-terminal deletion mutants in measurable assays of virologic function. We discovered that removal of up to 15 residues (IN 273) resulted in incremental diminution of enzymatic function and infectivity and that removal of the next three residues resulted in a loss of infectivity. However, replication competency was surprisingly reestablished with one further truncation, corresponding to IN 269 and coinciding with partial restoration of integration activity, but it was lost permanently for all truncations extending N terminal to this position. Our analyses of these replication-competent and -incompetent truncation mutants suggest potential roles for the IN CTD in precursor protein processing, reverse transcription, integration, and IN multimerization.The defining hallmarks of retroviruses are reverse transcription of the viral genomic information as encoded in polyadenylated RNA and the subsequent integration of the copied DNA genome into that of a host cell. The latter is an essential and irreversible event which is mediated by the catalytic activities of the viral integrase protein (IN), the recent target of successful chemotherapeutic intervention against HIV-1 infection (1). HIV-1 IN is a 288-amino-acid, 32-kDa protein that is cleaved from the C terminus of the Gag-Pol polyprotein (Pr160 Gag-Pol ) via viral proteolytic activity. The biochemical mechanisms that lead to retroviral integration, which have been extensively studied in vitro, are defined by two catalytically related and sequentially dependent steps (18) which may be distinguished by their respective sensitivities to current inhibitors of IN function (39). Following the completion of reverse transcription of the viral RNA into its DNA copy, IN removes two nucleotides from the 3Ј end of each strand of the viral DNA. This step, termed 3Ј processing, generates a chemically reactive 3Ј-hydroxyl group (CA OH -3Ј) at the 3Ј ends of the DNA molecule, effectively activating the termini for the subsequent reaction (strand transfer). This enzymatic step is the target of all I...

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen–host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.

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