A key step in the replication cycle of human immunodeficiency virus, type 1 (HIV-1) 2 involves the synthesis of doublestranded DNA copies of the diploid, single-stranded RNA genome by viral reverse transcriptase (RT) within the cytoplasm of infected cells. This newly synthesized viral DNA becomes an integral component of a large nucleoprotein complex termed the preintegration complex (PIC). PIC-associated proteins include HIV-1 matrix, Vpr, RT, and integrase (IN) (1-4), as well as cellular components such as lens epitheliumderived growth factor p75 (5), barrier-to-autointegration factor (6), and the high mobility group I(Y) protein (7). The PIC traverses the nuclear envelope, and within the nucleus the viral DNA is subsequently inserted into the chromosomes of the infected host, an event specifically catalyzed by IN.HIV-1 IN and RT (a heterodimeric enzyme of 66-and 51-kDa subunits) are initially synthesized as part of the Gag-Pol polyprotein, which is subsequently processed by HIV-1 protease during viral maturation to produce active IN and RT. IN (288 amino acids; 32 kDa) comprises three domains: an N-terminal zinc-binding domain (residues 1-50), a catalytic core domain (residues 51-212), and a C-terminal domain (CTD; residues 213-288) that binds DNA nonspecifically (reviewed in Ref. 8). IN has multiple effects throughout the viral life cycle, as perturbations within the IN coding sequence of the pol gene can impact not only integration but also PIC nuclear import, proper virion maturation, and reverse transcription (9 -20).Purified HIV-1 IN and RT have been found to physically interact in co-immunoprecipitation, glutathione S-transferasebased pulldown, and surface plasmon resonance (SPR) experiments (15,18,21,22), and assays with IN deletion constructs showed the IN CTD is both necessary and sufficient for this association (18,21). The significance of this IN-RT interaction in the context of viral replication has not yet been firmly established. In vitro studies with purified IN and RT, however, have shown that HIV-1 IN can stimulate both the initiation and elongation modes of RT-catalyzed reverse transcription by enhancing RT processivity (23).To aid our ongoing studies of functional interactions between IN and RT during HIV-1 replication, we have characterized in detail the RT-binding surface on the IN CTD using nuclear magnetic resonance (NMR) spectroscopy, identifying particular IN CTD amino acids that form contacts with RT through chemical shift perturbation experiments. In addition, we have assessed kinetic parameters and binding constants for both IN CTD-RT and IN-RT interactions using SPR. This experimental approach allows us to define and quantify the IN-RT interaction to a level that is not possible through coimmunoprecipitation or other fusion protein pulldown techniques.With the intention of perturbing IN-RT interactions, we introduced an alanine substitution for lysine at position 258 (K258A) within the IN CTD and subsequently used SPR to characterize the effect of this substitution upon IN⅐RT comp...

Human immunodeficiency virus type 1 (HIV-1T o establish an infection after entry into a susceptible cell, human immunodeficiency virus type 1 (HIV-1) has to reverse transcribe its RNA genome to double-stranded DNA, followed by integration into the host genome. Reverse transcriptase (RT) and integrase (IN) are the viral enzymes responsible for catalyzing the essential steps of reverse transcription and integration, respectively. Both enzymes are synthesized as part of the Gag-Pol polyprotein, which is later processed by the viral protease to produce active RT and IN during HIV-1 maturation (1, 2). RT is a heterodimeric enzyme consisting of 66-and 51-kDa subunits and catalyzes the RNA-and DNA-dependent reverse transcription of the viral RNA genome into double-stranded cDNA through a complex cascade of events (3, 4). The 32-kDa IN has three domains: an N-terminal zinc-binding domain, a catalytic core domain, and a C-terminal domain (CTD) that binds DNA nonspecifically. IN catalyzes the integration of the viral cDNA into the host genome in two steps: an initial 3=-end processing step that removes two nucleotides at each 3= end and exposes a highly conserved CA 5= overhang, followed by a strand transfer step that inserts both processed viral DNA ends into the host cell genome (5, 6). In vitro, IN can also catalyze a reverse reaction, termed disintegration, resolving a DNA mimic of the viral-host DNA intermediate to products corresponding to a 3= processed viral DNA end and a target duplex DNA (7). IN can multimerize and forms a complex with viral DNA ends, termed the intasome (8-10). Structural studies of the prototype foamy virus (PFV) intasome found the tetramer to be the active IN configuration (9,11,12). HIV-1 IN has also been proposed to function as a tetramer (10,(13)(14)(15)(16)(17).Mutations in

Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases.