Ubiquitin ligase Nedd4-2 modulates Kv1.3 current amplitude and ion channel protein targeting.

Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493-498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density.

Measurement of human immunodeficiency virus type 1 preintegration transcription by using Rev-dependent Rev-CEM cells reveals a sizable transcribing DNA population comparable to that from proviral templates.

Preintegration transcription is an early process in human immunodeficiency virus type 1 infection and has been suggested to occur at a low level. The templates have also been suggested to represent a small population of nonintegrated viral DNA, particularly the two-long-terminal-repeat (2-LTR) circles. However, these determinations were made by either using PCR amplification of viral transcripts in bulk cell populations or utilizing the LTR-driving reporter cells that measure the synthesis of Tat. The intrinsic leakiness of LTR often makes the measurement of low-level viral transcription inaccurate. Since preintegration transcription also generates Rev, to eliminate the nonspecificity associated with the use of LTR alone we have developed a novel Rev-dependent indicator cell, Rev-CEM, to measure preintegration transcription based on the amount of Rev generated. In this report, using Rev-CEM cells, we demonstrate that preintegration transcription occurs on a much larger scale than expected. The transcribing population derived from nonintegrated viral DNA was comparable (at approximately 70%) to that derived from provirus in a productive viral replication cycle. Nevertheless, each nonintegrated viral DNA template exhibited a significant reduction in the level of transcriptional activity in the absence of integration. We also performed flow cytometry sorting of infected cells to identify viral templates. Surprisingly, our results suggest that the majority of 2-LTR circles are not active in directing transcription. It is likely that the nonintegrated templates are from the predominant DNA species, such as the full-length, linear DNA. Our results also suggest that a nonintegrating lentiviral vector can be as effective as an integrating vector in directing gene expression in nondividing cells, with the proper choice of an internal promoter.

HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells.

Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here, we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction and that HIV utilizes the Galphai-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin-depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a postentry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.

Human macrophages support persistent transcription from unintegrated HIV-1 DNA.

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.