Yukie Iwabu

HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes.

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, β-transducin repeat-containing protein (βTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a βTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.

Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

A Novel Diagnostic Method for Human Immunodeficiency Virus Type-1 in Plasma by Near-Infrared Spectroscopy

Presently, the diagnosis of virus infections is based mainly on serological assays. Although polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) have been increasingly used for the diagnosis of such viral infections, the risk of transfusion‐transmitted blood‐borne viruses remains. Furthermore, PCR and ELISA are expensive and time‐consuming, and sometimes cause false‐positive or false‐negative results. Therefore, a rapid, accurate and cost‐effective diagnostic procedure is needed. We subjected plasma from individuals infected with human immunodeficiency virus type‐1 (HIV‐1), the causative agent of acquired immune deficiency syndrome (AIDS), as well as plasma from uninfected individuals as a control to near‐infrared (NIR) spectroscopy, which may provide a rapid diagnostic method for HIV‐1 infection without using any reagent. NIR spectra in the 600–1,000 nm region for plasma from pre‐serologically HIV‐1‐infected individuals and healthy donors were subjected to partial least squares (PLS) regression analysis and leave‐out cross‐validation to develop a multivariate model to estimate the concentration of HIV‐1. Simultaneously, the same plasma samples were examined for HIV‐1 p24 by ELISA. The results obtained by the NIR spectroscopy model for HIV‐1 yielded a good correlation with those obtained by the reference method (HIV‐1 p24 ELISA). These results suggest that NIR spectroscopy using plasma could provide a rapid, accurate, cost‐effective tool for large‐scale diagnosis of HIV‐1 infection.

Membrane-associated RING-CH (MARCH) 8 mediates the ubiquitination and lysosomal degradation of the transferrin receptor

Summary The transferrin receptor (TfR) mediates the uptake of transferrin (Tf)-bound iron from the plasma into the cells of peripheral tissues. The TfR continuously recycles between the plasma membrane and early/recycling endosomes. TfR expression is tightly controlled by the intracellular iron concentration through the regulation of TfR mRNA stability. However, much less is known about the mechanism by which TfR is degraded in cells. Previously, we reported a correlation between TfR ubiquitination and its iron-induced lysosomal degradation. The identification and characterization of a specific ubiquitin ligase for TfR is important in understanding the mechanism of iron homeostasis. Here, we show that membrane-associated RING-CH (MARCH) 8 ubiquitinates TfR and promotes its lysosomal degradation. Similar to other RING-type ubiquitin ligases, the RING-CH domain of MARCH8, which is located in the N-terminal cytoplasmic domain, is essential for the ubiquitination and downregulation of TfR. MARCH8 specifically recognizes the transmembrane domain of TfR and mediates ubiquitination of its cytoplasmic domain. In addition, the six-amino-acid sequence located in the C-terminal domain of MARCH8, which is highly conserved among different species, is required for the downregulation of TfR. Finally, and most importantly, TfR expression was markedly increased by siRNA-mediated knockdown of endogenous MARCH8. These findings demonstrate that the endogenous level of MARCH8 regulates TfR protein turnover through the downregulation and ubiquitination of TfR.

Structural Basis for the Antiviral Activity of BST-2/Tetherin and Its Viral Antagonism

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of HIV-1 and other enveloped viruses. In turn, these viruses have evolved specific antagonists to counteract this host antiviral molecule, such as the HIV-1 protein Vpu. BST-2 is a type II transmembrane protein with an unusual topology consisting of an N-terminal cytoplasmic tail (CT) followed by a single transmembrane (TM) domain, a coiled-coil extracellular (EC) domain, and a glycosylphosphatidylinositol (GPI) anchor at the C terminus. We and others showed that BST-2 restricts enveloped virus release by bridging the host and virion membranes with its two opposing membrane anchors and that deletion of either one completely abrogates antiviral activity. The EC domain also shows conserved structural properties that are required for antiviral function. It contains several destabilizing amino acids that confer the molecule with conformational flexibility to sustain the protein’s function as a virion tether, and three conserved cysteine residues that mediate homodimerization of BST-2, as well as acting as a molecular ruler that separates the membrane anchors. Conversely, the efficient release of virions is promoted by the HIV-1 Vpu protein and other viral antagonists. Our group and others provided evidence from mutational analyses indicating that Vpu antagonism of BST-2-mediated viral restriction requires a highly specific interaction of their mutual TM domains. This interpretation is further supported and expanded by the findings of the latest structural modeling studies showing that critical amino acids in a conserved helical face of these TM domains are required for Vpu–BST-2 interaction and antagonism. In this review, we summarize the current advances in our understanding of the structural basis for BST-2 antiviral function as well as BST-2-specific viral antagonism.

Superinfection of defective human immunodeficiency virus type 1 with different subtypes of wild-type virus efficiently produces infectious variants with the initial viral phenotypes by complementation followed by recombination.

Superinfection rates of human immunodeficiency virus type 1 (HIV-1) have increasingly been leading to more variation in HIV-1, as evidenced by the emergence of circulating recombinant forms (CRFs). We recently reported complementation in a persistently replication-defective subtype B-infected cell clone, L-2, by superinfection with CRF15_01B. The L-2 cells continuously produce immature particles due to a one-base insertion at pol protease. Proviruses in the superinfected cells carried both subtypes and produced particles with a mature morphology. In this study, we examined possible recombination following complementation to generate replication-competent variants by using three cell clones prepared from superinfected L-2 cells. The individual clones predominantly expressed the initial subtype B-derived mature Gag proteins. However, the viral particles carried both subtype B with the mutation and wild-type CRF15_01B at pol, suggesting the generation of virions with heterozygous RNAs. Interestingly, with cell-free passages of the progeny, defective particles disappeared, and were replaced with heterogeneous recombinants in the pol region with sequences derived from CRF15_01B that expressed subtype B phenotype. Thus, even a defective form of persistent HIV-1 can become replication-competent through superinfection-mediated complementation followed by recombination. These findings suggest the significance of long-lived infected cells as recipients for superinfection.

APOBEC3G oligomerization is associated with the inhibition of both Alu and LINE-1 retrotransposition.

Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A–H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

Sites of Action of HIV-1 Vpu in BST-2/Tetherin Downregulation

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of human immunodeficiency virus type 1 (HIV-1) by directly cross-linking virions to the membrane of infected cells. This antiviral effect is counteracted by the HIV-1 accessory protein viral protein U (Vpu) through mechanisms that remain unclear. Accumulating evidence suggests that Vpu antagonizes BST-2 by removing it from the plasma membrane; however, neither the cellular sites of interaction nor the effector mechanisms that result in the downregulation of BST-2 cell-surface expression have been fully determined. Based on current evidence regarding the subcellular localization of Vpu and BST-2 and the latters trafficking defects induced by their interaction, three models have been proposed. In the first, Vpu is hypothesized to block the traffic of newly synthesized BST-2 towards the cell surface by retaining it in the biosynthetic/secretory compartment. The second model suggests that Vpu sequesters BST-2 within intracellular compartments corresponding to recycling endosomes and the trans-Golgi network by blocking its recycling after endocytosis. In the third model, we and others have proposed that Vpu directly internalizes BST-2 from the plasma membrane and induces its enhanced endolysosomal trafficking and degradation. As for its intracellular fate, the viral antagonism of BST-2 is likely dependent on the intracellular sequestration, or the proteasomal/lysosomal degradation of the restriction factor. This review summarizes the current advances in our understanding of the cellular pathways and sites of action of Vpu in the downregulation of cell-surface BST-2.

Direct internalization of cell-surface BST-2/tetherin by the HIV-1 accessory protein Vpu

The host transmembrane protein BST-2/tetherin is a powerful antiviral factor that blocks the production of enveloped viruses. The HIV-1 accessory protein Vpu inhibits the antiviral activity of BST-2; however, the degradation pathway by which Vpu downregulates BST-2 from the cell surface and the actual subcellular location where Vpu targets BST-2 for downregulation remain controversial. Whereas one study showed that Vpu acts on constitutively endocytosed BST-2, we recently reported that Vpu can internalize BST-2 from the cell surface. Because the evidence for this conclusion was derived from indirect results, we present direct evidence in this study using an antibody internalization assay with an endocytosis-defective mutant of BST-2. The internalization of the BST-2 protein into cells coexpressing wild-type Vpu was observed when the cells were preincubated with antibodies against BST-2 at 37˚C, but not at 4˚C, for 10 min. These results strongly support our previous finding that continuously expressed de novo BST-2 at the cell surface is internalized by functional Vpu protein.

Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2alpha. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2alpha. Confocal fluorescence microscopy revealed that a subpopulation of AP2alpha was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin beta and Nup153, implying that AP2alpha negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2alpha may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.