ABSTRACT
The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. The power of this method to investigate viral events occurring between the cytoplasmic and the nuclear compartment is further shown in this study through the analysis of HIV-IN-EGFP in cells expressing the TRIMCyp restriction factor. In these cells the HIV-IN-EGFP complexes are not detected in the nuclear compartment, while treatment with MG132 reveals an accumulation of HIV-1 complexes in the cytoplasm. However, the Vpr-mediated transincorporation strategy used to incorporate IN fused to EGFP (IN-EGFP) impaired viral infectivity. To optimize the infectivity of the HIV-IN-EGFP, we used mutated forms of IN (E11K and K186E) known to stabilize the IN complexes and to partially restore viral infectivity in transcomplementation experiments. The fluorescent particles produced with the modified IN [HIV-IN(K)EGFP_IN(E)] show almost 30% infectivity as compared to wild-type NL4.3. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.
Subject(s)
Cell Nucleus/virology , HIV Infections/genetics , HIV-1/physiology , Virus Internalization , Cell Line, Tumor , Fluorescent Dyes/analysis , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , HIV Infections/virology , HIV-1/genetics , HeLa Cells , Humans , Microscopy, Fluorescence/methods , Virus Integration/genetics , Virus Replication/geneticsABSTRACT
RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.
Subject(s)
Biotin/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group C/chemistry , Protein Interaction Mapping , Proteome/analysis , Amino Acid Sequence , Biological Assay , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , ELAV Proteins/chemistry , ELAV Proteins/genetics , ELAV Proteins/metabolism , ELAV-Like Protein 1 , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , Humans , Molecular Sequence Data , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Poly(A)-Binding Protein I/chemistry , Poly(A)-Binding Protein I/genetics , Poly(A)-Binding Protein I/metabolism , Protein Binding , Protein Biosynthesis , Protein Interaction Maps , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism , Streptavidin/chemistryABSTRACT
In many countries, HIV testing among tuberculosis (TB) patients is recommended so that both infections are appropriately treated. Cross-reacting antibodies to HIV antigens have been reported for several conditions, including TB, leprosy, malaria, and rheumatoid arthritis. To study the pattern and prevalence of cross-reacting antibodies to HIV antigens, we examined sera from 153 HIV-negative TB patients and 40 healthy individuals in Chennai, south India. We also studied the differences in cross-reactivity of various HIV antigens using two different Western blot kits. Of the 153 samples studied, 80 were tested using HIV Western blot and 73 were tested using INNOLIA. Most patients in the study had concordantly negative ELISA and rapid tests, and no subject had a positive Western blot. However, seven TB patients had antibodies that cross-reacted with HIV antigens, giving rise to an indeterminate result. While p51/55 was the most frequently recognized antigen in the Western blot assay, antibodies to sgp120 was most frequently identified in INNOLIA. Sequence similarities between the two organisms could be responsible for eliciting cross-reacting antibodies, since a few related epitopes were identified in HIV and Mycobacterium. These findings could have potential implications for the development of diagnostics and vaccines.
