ABSTRACT
Members of the serine incorporator (SERINC) protein family exert broad antiviral activity, and many viruses encode SERINC antagonists to circumvent these restrictions. Significant new insight was recently gained into the mechanisms that mediate restriction and antagonism. In this review, we summarize our current understanding of the mode of action and relevance of SERINC proteins in HIV-1 infection. Particular focus will be placed on recent findings that provided important new mechanistic insights into the restriction of HIV-1 virion infectivity, including the discovery of SERINC's lipid scramblase activity and its antagonism by the HIV-1 pathogenesis factor Nef. We also discuss the identification and implications of several additional antiviral activities by which SERINC proteins enhance pro-inflammatory signaling and reduce viral gene expression in myeloid cells. SERINC proteins emerge as versatile and multifunctional regulators of cell-intrinsic immunity against HIV-1 infection.
Subject(s)
HIV Infections , Membrane Proteins , Humans , Membrane Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Host-Pathogen Interactions , Virion/metabolism , Antiviral AgentsABSTRACT
In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.
Subject(s)
Protozoan Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/metabolism , 5' Untranslated Regions , Base Sequence , Polyribosomes/metabolism , Protein Binding , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , RNA Interference , RNA, Double-Stranded/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Transcriptome , Zinc FingersABSTRACT
The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.
Subject(s)
Isotope Labeling/methods , Polyribosomes/metabolism , Protein Biosynthesis , Trypanosoma brucei brucei/genetics , Amino Acids/chemistry , Amino Acids/metabolism , Centrifugation, Density Gradient/methods , Metabolomics/methods , Parasitology/methods , Polyribosomes/chemistry , RNA, Messenger/chemistry , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , RNA, Protozoan/chemistry , RNA, Protozoan/isolation & purification , RNA, Protozoan/metabolism , Sulfur Radioisotopes/chemistry , Trypanosoma brucei brucei/metabolismABSTRACT
Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We show here that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Pull-down analysis of tagged ERBP1 suggests that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.
