A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein.

The interferon-induced transmembrane (IFITM) proteins have been recently shown to restrict HIV-1 and other viruses. Here, we provide evidence that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env), thereby inhibiting viral infection. IFITM proteins interact with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions does not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes leads to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appears to be virus-strain dependent. Overall, our study uncovers a mechanism by which IFITM proteins specifically antagonize HIV-1 Env to restrict HIV-1 infection and provides insight into the specialized role of IFITMs in HIV infection.

P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. IMPORTANCE: This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a stepwise determination of when these compounds inhibit HIV-1 infection. These data provide a rationale for the development of novel antiretroviral therapies that have a dual role in both direct antiviral activity and the reduction of HIV-associated inflammation. Purinergic antagonists are shown here to have equivalent efficacy in inhibiting HIV infection via cell-free and cell-to-cell infection, and it is shown that purinergic receptors could provide an attractive therapeutic anti-HIV target that might avoid resistance by targeting a host signaling pathway that potently regulates HIV infection. The high-throughput screen of HIV-1 fusion inhibitors further defines P2X-selective compounds among the purinergic compounds as being the most potent HIV entry inhibitors. Clinical studies on these drugs for other inflammatory indications suggest that they are safe, and thus, if developed for use as anti-HIV agents, they could reduce both HIV replication and HIV-related inflammation.

In vivo [35S]-methionine incorporation.

The purpose of this assay is to measure the incorporation of radiolabeled [(35)S]-methionine into newly synthesized proteins in exponentially growing yeast cells. This allows for a quantitative in vivo measurement of total protein synthesis.

Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis.

Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.

The eukaryotic translation elongation Factor 1Bgamma has a non-guanine nucleotide exchange factor role in protein metabolism.

The turnover of damaged proteins is critical to cell survival during stressful conditions such as heat shock or oxidative stress. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is toxic to cells. Therefore these proteins must be efficiently exported from the ER and degraded by the proteasome or the vacuole. Previously it was shown that the loss of eukaryotic elongation factor 1Bγ (eEF1Bγ) from the yeast Saccharomyces cerevisiae results in resistance to oxidative stress. Strains lacking eEF1Bγ show severe defects in protein turnover during conditions of oxidative stress. Furthermore, these strains accumulate a greater amount of oxidized proteins, which correlates with changes in heat shock chaperones. These strains show severe defects in vacuole morphology and defects related to the maturation of carboxypeptidase Y that is not dependent on the catalytic subunit of the eEF1B complex as a guanine nucleotide exchange factor. Finally, eEF1Bγ co-immunoprecipitates with an essential component of ER-Golgi transport vesicles. Taken together, these results support a broader protein metabolism role for eEF1Bγ.

Eukaryotic polyribosome profile analysis.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.

Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor.

Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3' end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.

A birth-to-death view of mRNA from the RNA recognition motif perspective.

RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process.