Virology Journal Club 21.02.2013

Nucleolus This term students taking my virology course at the University of Leicester are doing a series of tutorials involving reading and explaining research papers in virology. This is the sort of exercise which is common in graduate schools and regularly performed by researchers, and is known as a “journal club”. We cannot give students access to the facilities or equipment to study dangerous human viruses at the forefront of research, so gaining an deep understanding of the way into which research is currently being conducted in this area is the closest we can come to allowing them to be “real virologists”. Today, we are looking at the following paper:

Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus (2012) PLoS ONE 7(11): e48702

 

How do you read a research paper? Start with a quick scan: what’s this all about?

From the Abstract:

  • The HIV-1 Tat protein is trans-activator of gene expression essential for HIV-1 replication.
  • Tat trafficks to the nucleoplasm and the nucleolus.
  • The nucleolus is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection.
  • Investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. [What is TAP? Why did they use this?]
  • Organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), used to quantify 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus on Tat expression.
  • Spatial control and subcellular compartmentalisation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. [Pathogenesis? Do they mean replication?]
  • Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signalling pathways and genome integrity. [Lots of stuff then - non-specific effect?]

At this stage it is useful to scan though the figures and explanatory figure legends in the paper to get an overview of the data:

Figure 1: Proteomic workflow and validation studies
Transfected Jurkat T cells with a vector expression HIV-1 Tat, and also control cells (expression baseline), with the same vector without Tat. Confirmed expression of active Tat protein by luciferase assay. Isolated nucleola. Subcellular fractionation checked by Western-Blot using anti-nucleolin (nucleolar), anti-fibrillarin (nucleolar), anti-α-tubulin (cytoplasmic) and anti-PARP (nuclear) antibodies. Nucleolar location of Tat conformed by fluorescent microscopy. Protein expression examined by MS, MaxQuant (MS analysis software), Western blot confirmation, relative abundance is plotted – green shows depletion, red enrichment.

Figure 2: 49 nucleolar proteins showing a 1.5 fold or greater significant change (p<0.05) on Tat expression.

Figure 3: Western-Blot results comparing expression levels of selected 15 proteins
[Selected how – why these 15?]

Figure 4: Network analysis of the Tat interactome
Panel B: HIV-1 Tat interacts with 146 proteins out of the 416 proteins identified in the mixed nucleolar fraction.

Figure 5: Overall profiles of changes in protein abundance in the nucleolus of T-cells on HIV-1 Tat expression
Gene ontology [relationships] (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (red = upregulated, green = downregulated):

  • Glycolysis & pentose phosphate pathway
  • Nucleotide metabolism
  • Ubiquitin proteasome pathway
  • Molecular chaperones
  • DNA replication & repair
  • Splicesomes
  • rRNA processing
  • Ribosomal proteins

Analysis checklist:

1. What did the authors want to find out or prove? Why? (Introduction section of the paper)
The nucleolus is a non-membrane bound structure within the nucleus of eukaryotic cells. Its main function is to transcribe ribosomal RNA (rRNA) and assemble it into ribosomes. A number of viruses interact with the nucleolus during replication, e.g: HIV-1, hCMV, HSV and KSHV. HIV-1 Tat was know to accumulate in the nucleolus. The authors wanted to investigate the overall pattern of nucleolar protein expression (proteome) on Tat expression.

2. What exactly did they do? (Methods section)
The study was performed in the Jurkat T cell line (1977). Cells were transfected using a VSV pseudotyping method (see: Lentiviral Vectors). Cells were labelled with stable (non-radioactive) heavy isotopes and subcellular fractions prepared. Proteins were identified and measured by mass spectroscopy.

3. What were the results? (Results section)
Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry.
This allows investigation of protein interactions under “natural” physiological conditions. The concentrations of 520 nucleolar proteins were measured by mass spectroscopy. By comparing cells expression tat with those transfected with the vector alone (control), 49 nucleolar proteins were found to show a 1.5 fold or greater significant change (p<0.05) on Tat expression. Long discussion of what these proteins do. A total of 5060 interactions were found. What does this all mean???

4. What do these results mean? (Discussion section)
Biomolecular Pathway Dysregulation by HIV-1 Tat – but so what? In several places the authors refer to “HIV-1 pathogenesis” – but what do these results tell us about pathogenesis?

5. What else could the authors have done? What should they do next? What are the strengths and weaknesses of this paper? Why does this research matter? (Synthesis)
This is a poorly written paper – contains typos, badly expressed sentences.

Discussion: “The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1″ – what does this mean? The references cited here refer to proteomic studies.

“Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production.” What does this filler sentence actually mean?

So what? “Omics” – is this just a fishing trip? What about hypothesis-driven research?

Why was this paper published in PLOS One and not another journal? How are papers refereed for PLOS One? How many times has this paper been cited? How many times do most papers get cited?

“While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression.”
This is a significant advance in knowledge.

 

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