HIV-1 integrase (IN) is

HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin ␣ (Imp␣) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Imp␣3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4؉ C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Imp␣3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Imp␣3 in HIV nuclear import. In an attempt to understand how Imp␣3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Imp␣3 both in a 293T cell expression system and in HIV-infected CD4 ؉ C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Imp␣3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.HIV-1 replicates productively in nondividing cells, such as monocytes (49,61,74), macrophages (23,37,59,65,71), dendritic cells (47,64), and resting CD4 ϩ T lymphocytes (86), through its ability to undergo active nuclear import by hijacking the host nuclear import machinery. Moreover, active nuclear import is not only required for nondividing-cell infection but also plays a role in the infection of proliferating cells (35). This ability of HIV-1 to enter the nucleus at interphase may contribute significantly to the very high replication rate observed in infected individuals (30,70,73) and is one of the crucial steps in HIV-1 replication, which plays a leading role in the establishment of infection and AIDS pathogenesis.The viral double-stranded DNA (dsDNA), which associates with viral and cellular proteins, forms a high-molecular-mass nucleoprotein complex called the preintegration complex (PIC) in the cytosol of an infected cell (15,51). This large complex has to actively enter the nucleus through the intact nuclear membrane in order to be integrated. At the molecular level, the active nuclear import ability of HIV-1 is attributed to the karyophilic properties of viral PICs. It is known that several viral nucleophilic proteins, including integrase (IN), matrix (MA), and Vpr, are associated with this nucleoprotein complex and pla...

De novo resistance and rapid recurrence often characterize responses of B-cell malignancies to ibrutinib (IBR), indicating a need to develop drug combinations that block compensatory survival signaling and give deeper, more durable responses. To identify such combinations, we previously performed a combinatorial drug screen and identified the Bcl-2 inhibitor venetoclax (VEN) as a promising partner for combination with IBR in Mantle Cell Lymphoma (MCL). We have opened a multi-institutional clinical trial to test this combination. However, analysis of primary samples from patients with MCL as well as chronic lymphocytic leukemia (CLL) revealed unexpected heterogeneous de novo resistance even to the IBR+VEN combination. In the current study, we demonstrate that resistance to the combination can be generated by microenvironmental agonists: IL-10, CD40L and, most potently, CpG-oligodeoxynucleotides (CpG-ODN), which is a surrogate for unmethylated DNA and a specific agonist for TLR9 signaling. Incubation with these agonists caused robust activation of NF-κB signaling, especially alternative NF-κB, which led to enhanced expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, thus decreasing dependence on Bcl-2. Inhibitors of NF-κB signaling blocked overexpression of these anti-apoptotic proteins and overcame resistance. Inhibitors of Mcl-1, Bcl-xL, or survivin also overcame this resistance, and showed synergistic benefit with the IBR+VEN combination. We conclude that microenvironmental factors, particularly the TLR9 agonist, can generate de novo resistance to the IBR+VEN combination in CLL and MCL cells. This signaling pathway presents targets for overcoming drug resistance induced by extrinsic microenvironmental factors in diverse B-cell malignancies.

In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150 Glued T he steps of early stage human immunodeficiency virus type 1 (HIV-1) replication include virus entry, uncoating, reverse transcription, intracytoplasmic retrograde transportation (i.e., the migration of HIV from the cytoplasmic periphery to the perinuclear space), nuclear import, and genomic integration (reviewed in reference 1). Following HIV-1 entry into the cell, viral genomic RNA and associated proteins are released into the cytoplasm as a ribonucleoprotein complex referred as the reverse transcription complex (RTC). Within the RTC, HIV-1 genomic RNA is reverse transcribed into a cDNA, which then forms a highmolecular-weight preintegration complex (PIC). HIV-1 cDNA enters the nucleus as a part of PIC by active nuclear import and subsequently integrates into the host cell genome (reviewed in reference 2).HIV-1 utilizes various cellular proteins for replication mostly by interacting with its viral proteins. Genome-wide small interfering RNA (siRNA)/short hairpin RNA (shRNA) screening as well as other functional studies have uncovered a large number of host proteins with putative roles in HIV-1 replication (reviewed in references 3, 4, and 5). Additionally, functional studies from our laboratory, as well as from other groups, have uncovered key viral and cellular protein interactions that promote successful HIV-1 nuclear import and integration (reviewed in references 2 and 6). However, molecular events associated with HIV-1 reverse transcription, uncoating, or retrograde transport in the cytoplasm are not well understood. To date, evidence suggests that gem-associated protein 2 (Gemin2) interacts with HIV-1 integrase (IN) in target cells and contributes to reverse transcription by an unknown mechanism(s) (7,8). Similarly, accumulated evidence suggests that cyclophilin A (CypA) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (prolyl isomerase Pin1) proteins interact with HIV-1 capsid (CA) protein in target cells and facilitate the proper uncoating of HIV-1 (9, 10). In addition, some other cellular factors with putative roles in HIV-1 reverse transcription and uncoating have been described in recent studies (11)(12)(13)(14). Although the exact mechanism(s) by which these cellular factors contribute to HIV-1 reverse transcription and/or uncoating is not very clear, the accumulated evidence so far clearly suggests a key role for cellular cofactors in HIV-1 uncoating and reverse transcription.Dynein adapter proteins such as dynein light chain 1 (DYNLL1, LC8, DLC1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued have been implicated in cargo recruitment to the dynein complex during retrograde transport (15-18). The dynein complex is a microtubule (MT)-associated protein c...