免疫学技术

Along with CD4+ T-lymphocytes, macrophage lineage cells serve as primary hosts for HIV replication in vivo. In some tissues such as brain, where T-cell infection is essentially absent, the development of HIV-associated disease is mediated through infection of macrophages. This fact und ...

Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly the β-chemokine receptor 5 (CCR5) and the α-chemokine receptor 4 (CXCR4). Viral isolates can be phenotypically classified based on the co-recep ...

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary ...

The requirement of HIV-1 and HTLV-1 to export incompletely spliced mRNAs has necessitated the evolution of Rev and Rex, respectively, to overcome host cellular mechanism that block nuclear-cytoplasmic export of incompletely processed mRNAs. Evaluating the function of these viral f ...

Upon integration into the host cell genome, the nucleosomal organization and epigenetic control of the HIV-1 provirus play an active role in its transcriptional regulation. Therefore, characterization of the chromatin changes that occur in the viral promoter region in response to dif ...

The Ribonuclease (RNase) H is one of the four enzymes encoded by all retroviruses, including HIV. Its main activity is the hydrolysis of the RNA moiety in RNA–DNA hybrids. The RNase H ribonuclease is essential in the retroviral life cycle, since it generates and removes primers needed by the Reverse Tr ...

Reverse transcription is an obligatory step in retrovirus replication in the course of which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded positive sense RNA genome to synthesize the double-stranded viral DNA. At the same time the RT-associated RN ...

Advancements in fluorescent microscopy techniques now permit investigation of HIV-1 biology exploiting tools alternative to conventional molecular biology. Here we describe a novel, fluorescence-based method to visualize HIV-1 viral particles within intact nuclei of inf ...

Uncoating is an early step of HIV-1 replication in which the viral capsid disassembles by p24 capsid (p24CA) protein dissociating from the viral complex. Although uncoating is required for HIV-1 replication, many questions remain about the mechanism of this process as well as its impact on oth ...

The uncoating process of HIV-1 is a poorly understood process, so the development of a reliable assay to study uncoating is critical for moving the field forward. Here we describe an uncoating assay that currently represents the state-of-the-art biochemical procedure for monitoring unco ...

After virus fusion with a target cell, the viral core is released into the host cell cytoplasm and undergoes a controlled disassembly process, termed uncoating, before or as reverse transcription takes place. The cellular protein TRIM5α is a host cell restriction factor that blocks HIV-1 inf ...

The HIV-1 entry receptors are CD4 and a chemokine receptor (CCR5 or CXCR4). In addition it has recently been demonstrated that HIV-1 gp120 binds to and signals through integrin α4β7, the gut-homing receptor (Arthos et al., Nat Immunol 9(3):301–309, 2008). Integrin α4β7 is not an entry receptor for HIV-1, ...

Assembly, release and maturation of HIV-1 particles comprise a highly dynamic sequence of events, characterized by a series of dramatic rearrangements of the viral structural proteins and overall virion architecture. HIV-1 morphogenesis is a relatively rapid and asynchronous pr ...

Vpx is coded almost exclusively by members of the SIVSM/HIV-2 lineage of primate lentiviruses, it is incorporated into virion particles and is thus present during the early phases of infection of target cells. While Vpx exerts no detectable function during the infection of most cell types, it po ...

HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by fu ...

Nef is a multifunctional protein encoded by all primate lentiviruses that modulates cell surface expression of a variety of cellular receptors and increases the infectivity of progeny virons. Here, we describe the use of bicistronic HIV-1 constructs that coexpress Nef and fluorescent ...

HIV-1 viral protein R (VpR) is a multifunctional protein that plays specific roles at multiple stages of the HIV-1 viral life cycle and affects anti-HIV functions of the immune cells. VpR is required for efficient viral replication in nondividing cells such as macrophages, and it promotes, to some ...

Nucleocytoplasmic trafficking of proteins plays important roles in processes of the viral life cycle. Interspecies heterokaryon assay is one of the most effective methods to investigate the nucleocytoplasmic trafficking properties of a protein. In our lab, the interspecies het ...

High-throughput methods for screening of physical and functional interactions now provide the means to study virus–host interactions on a genome scale. The limited coverage of these methods and the large size and uncertain quality of the identified interaction sets, however, require ...

Many pathogens, including viruses, bacteria, as well as bacterial toxins, enter their target cells by endocytosis leading to accumulation of pathogenic and cellular proteins in endosomes. Here, we present detailed experimental instructions on isolation of endosomes after virus i ...