Nuclear Localization of HIV Type 1 Vif Isolated from a Long-Term Asymptomatic Individual and Potential Role in Virus Attenuation
ABSTRACT Recent reports have determined that HIV-1 Vif counteracts an innate antiviral cellular factor, Apobec3G. However, the function of Vif during HIV-1 pathogenesis remains poorly understood. To gain a better understanding of Vif function, the viral isolate from an HIV-1-infected long-term nonprogressor (LTNP) that displayed a Vif-mutant replication phenotype was studied. This LTNP has been infected since before 1983 and has no HIV-related disease in the absence of antiretroviral therapy. From separate samples, obtained on more than one study visit, virus grew in cocultures of LTNP cells with Vif-complementing T cell lines, but not the parental T cell lines. An unusual amino acid motif (KKRK) was found in the Vif sequence at positions 90 to 93. Since this motif commonly functions as a nuclear localization sequence, experiments were performed to determine the ability of this KKRK motif to mediate nuclear localization of Vif. Wild-type Vif displayed a predominantly cytoplasmic distribution. In contrast, the KKRK Vif showed a predominantly nuclear localization. The effect of the KKRK mutation on virus production and infectivity was also studied. The KKRK motif that mislocalizes Vif to the nucleus also reduces viral replication and infectivity in nonpermissive cells. Our data highlight the importance of Vif in HIV-1 pathogenesis and also provide a unique tool to investigate the interaction of Vif and Apobec3G.
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ABSTRACT: Human Immunodeficiency Virus Type 1 (HIV-1), the virus that causes Acquired Immunodeficiency Syndrome (AIDS), attacks the immune system leaving patients susceptible to opportunistic infections that eventually cause death. Highly Active Antiretroviral Therapy, HAART, is the current drug strategy used to combat HIV. It is a combination therapy that includes HIV-1 Reverse Transcriptase and HIV-1 Protease inhibitors. Drug resistant strains arise that evade current HAART treatments; therefore novel drugs are needed. HIV-1 regulatory proteins such as Tat, Rev, Nef, Vpr, Vpu, and Vif are attractive new drug targets. Of particular interest is the HIV-1 Vif protein and its cellular binding partner APOBEC3G. In the absence of HIV-1 Vif, APOBEC3G, a cytidine deaminase, is able to mutate the viral cDNA and render the virus noninfectious. HIV-1 Vif binds to APOBEC3G and targets it for proteosomal degradation through an interaction with a Cullin-RING ligase complex. Blocking the HIV-1 Vif APOBEC3G interaction would allow APOBEC3G to perform its antiviral function. An attractive strategy to target the HIV-1 Vif APOBEC3G interaction would be a structure-based one. To apply structure-based drug design approaches to HIV-1 Vif and APOBEC3G, I attempted to collect high resolution structural data on HIV-1 Vif and APOBEC3G. My attempts were unsuccessful because the milligram quantities of soluble protein required were not obtained. Therefore, in Chapter III I used chemical cross-linking and mass spectrometry to probe the structural topology of HIV-1 Vif obtaining low resolution structural data. Chemical cross-linking formed HIV-1 Vif multimers including dimers, trimers, and tetramers. Analysis of the cross-linked monomer revealed that HIV-1 Vif’s N-terminal domain is a well-folded, compact, globular domain, where as the C-teriminal domain is predicted to be disordered. In addition, disorder prediction programs predicted the C-terminal domain of HIV-1 Vif to be disordered. Upon oligomerization the C-terminal domain undergoes a disorder-to-order transition that not only facilitates oligomerization but may facilitate other protein-protein interactions. In addition, HIV-1 Vif oligomerization bring Lys34 and Glu134 in close proximity to each other likely creating one molecular surface forming a “hot spot” of biological activity. In Chapter IV I confirmed my low resolution structural data via peptide competition experiments where I identified peptides that can be used as scaffolds for future drug design. HIV-1 Vif oligomerization is concentration dependent. The HIV-1 Vif peptides Vif(29-43) and Vif(125-139) were able to disrupt HIV-1 Vif oligomerization, which confirms the low resolution structural data. HIV-1 Vif peptides Vif(25-39) and Vif(29-43) reduced the amount of APOBEC3G immobilized on the Protein A beads, reduced the amount of HIV-1 Vif interacting with APOBEC3G, or degraded APOBEC3G itself. These peptides could be used as scaffolds to design novel drugs that disrupt the function of HIV-1 Vif and or APOBEC3G. Therefore, low resolution structural data and peptide competition experiments were successful in identifying structurally important domains in HIV-1 Vif. They also provided insight into a possible mechanism for HIV-1 Vif function where a disorder-to-order transition facilitates HIV-1 Vif’s ability to interact with a diverse set of macromolecules. These data advance our structural understanding of HIV-1 Vif and they will facilitate future highresolution studies and novel drug designs.
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ABSTRACT: High-speed network traffic prediction is considered as the core of the preventive congestion control. In this paper, we apply two different artificial neural network (ANN) architectures, multilayer perceptron (MLP) and fuzzy neural network (FNN), to predict one-step ahead the value of the MPEG and JPEG video, Ethernet and Internet traffic data. To enhance prediction accuracy, the output of the individual ANN predictors are combined using different combination schemes. An adaptive updating scheme is used in both of the ANNs and combination schemes. This adaptation makes the predictors dynamic and allows them to capture the non-stationary traffic characteristics. Prediction at different time scales is considered in order to apply the predicted values to the congestion control schemes. The results show that the ANN predictors outperform the autoregressive (AR) model, and the combination approach enhances the prediction accuracy.Neural Networks, 2003. Proceedings of the International Joint Conference on; 08/2003
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ABSTRACT: Infection with HIV-1 leads to progressive CD4 T-cell death, resulting in AIDS development. The mechanisms that trigger this CD4 T-cell death are still not fully understood, but a lot of data indicates that apoptosis plays a major role in this cell demise. Both infected and uninfected CD4 T-cells can die during HIV-1 infection by different cell-death pathways, but HIV-1-induced, bystander, CD4 T-cell killing is now recognized as central to immunodeficiency. The HIV-1 directly modulates CD4 T-cell death using multiple different strategies in which several viral proteins have an essential role. Recent data demonstrate that relationships can exist between the three main types of programmed cell death, i.e. apoptosis, autophagic programmed cell death, and necrosis-like programmed cell death. Almost nothing is currently known about the role of necrosis-like programmed cell death in CD4 T-cell death induced by the viral proteins, but a very recent study demonstrates that autophagy is needed to trigger apoptosis of bystander CD4 T-cells, further increasing the level of complexity of this pathology. This review presents an overview of the major types of programmed cell death and details the mechanisms by which the HIV-1 viral proteins control both infected and uninfected CD4 T-cell death.AIDS reviews 10/2006; 8(4):221-36. · 3.79 Impact Factor