Why would you want to study proteins associated with viruses or virus infection? Receptors Mechanism of uncoating How is gene expression carried out, exclusively by viral enzymes? Gene expression phases? Virus encoded proteins offer potential targets for chemotheraputic agents. Where and how does assembly occur? Events involved in putting together complex particles are not understood, but probably involve interactions with the cytoskeleton and other host protein complexes Inclusions are often formed in the cytoplasm, what are these, what function do they have? How does virus accomplish cell to cell spread? Proteins as antigens Incorporation of host proteins as immune evasion strategy Structural vs. nonstructural proteins A. Structural viral proteins virion A C Incorporated in the virion particle B B. Nonstructural viral proteins cell Expressed in infected cells, but not incorporated into the virion particle C. Host proteins Specifically incorporated, passively incorporated?
Virus Proteomes: Identification of Proteins in Virus Particles A model: Washburn et al. identified 1,484 proteins in the yeast proteome using gel free liquid chromatography and tandem mass spectroscopy (LC/MS/MS) (Washburn, M. P., D. Wolters, and J. R. Yates III. 2001. Large scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242 247) Similar analyses have been used to identify virion proteins in Human cytomegalovirus (J. Virol. 78:10960 10966) Murine cytomegalovirus (J. Virol. 78:11187 11197) Epstein Barr virus (Proc. Natl. Acad. Sci. USA 101:16286 16291) Kaposi s sarcoma associated herpesvirus (J. Virol. 79:800 811) Previous approach: SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) Limited by gel resolution Limitations The majority of viral genes encode proteins smaller than 50 kda that tend to cluster together and are difficult to resolve by SDS PAGE N terminal sequencing of viral protein bands on gels shows that multiple protein bands can be derived from a single viral gene as a result of posttranslational cleavage or modification In many cases, less abundant virion components are detected but cannot be identified
Mass spectrometry (MS), in particular, tandem MS (MS/MS), provides a powerful tool for viral proteome analysis Much more sensitive than other methods Can deal with protein mixtures Offers a high throughput Vaccinia Virus (VV) assemblymultiple forms of virions: IV, IEV, IMV, CEV, EEV VV assembly is a complex process More than one hundred proteins participate Need to understand, at the molecular and cellular levels, how viral membranes and cores are formed, and what are the viral proteins involved in these events that lead to virion assembly and generation of infectious forms Process might also provide important insights in cell biology
Vaccinia virus particles are quite complex www.microbiologybytes.com Oval or "brick shaped" particles 200 400 nm long can be visualized by the best light microscopes. The external surface is ridged in parallel rows, sometimes arranged helically. The particles are extremely complex, containing many proteins (more than 100) and detailed structure is not known. Thin sections in E.M. reveal that the outer surface is composed of lipid and protein which surrounds the core, which is biconcave (dumbbell shaped), with two "lateral bodies" (function unknown). The extracellular forms contain 2 membranes (EEV extracellular enveloped virions), intracellular particles only have an inner membrane (IMV intracellular mature virions). Isolated VV particles (IMV) preserved by rapid freezing and viewed by cryo-em Cyrklaff M. et.al. PNAS 2005;102:2772-2777 2005 by National Academy of Sciences
Typical proteomics approach: Vaccinia IMV J Virol 80: 2127 2140 FIG. 1. (A) Purified IMV. Electron microscopy of purified IMV particles using negative uranyl acetate staining (left) and silver staining of IMV proteins, 340 ng (lane 1) and 170 ng (lane 2), on 12% SDS PAGE (right). (B) MS/MS spectrum of one tryptic peptide with a sequence identified as HAFDAPTLYVK and with a Mascot score of 72. (C) Amino acid sequence of the putative E6R protein. Tryptic peptides detected by MS, including the peptide in panel B, are underlined and give 53% sequence coverage. (D) MS/MS spectrum of one tryptic peptide with a sequence identified as ADEDDNEETLK and with a Mascot score of 80. (E) Amino acid sequence of A27L envelope protein. Tryptic peptides detected by MS, including the peptide in panel D, are underlined and give 71% sequence coverage. Human CMV, a complex virion structure University of Birmingham, UK www.wmin.ac.uk HCMV virion is composed of an icosahedral capsid that contains a linear 230 kbp double stranded DNA genome with attached proteins and an outer layer of proteins called tegument, surrounded by a cellular lipid layer containing viral glycoproteins
SUMMARY I: Virus proteins Identified 71 HCMV encoded proteins (double the number previously identified) Included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions 12 proteins from novel viral ORFs HCMV may express as many as 200 proteins (there are >200 potential ORFs) at various points in its life cycle, all proteins may not all be present at the same time SUMMARY II: : Host cellular proteins Identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones Some host cellular proteins were as abundant as viral proteins One of the host cell proteins pointed to sites in the cell where viruses are assembled Prevalence of host proteins in the virus, might also suggest how the virus avoids detection by the immune system
Role of viral promoter binding proteins and identifying viral/host protein binding partners Characterization of the relationships between promoters and transcription factors is significant to revealing the mechanisms involved in: gene regulation cellular differentiation cellular susceptibility to viral infection (viral tropism) Goal: Identification of transcription factors, transcription factor complexes, and activation states of transcription factors binding a promoter of interest Differential comparison of transcription factor binding between various cell types, and at various stages of cellular maturity and/or cellular activation Comparison of transcription factor binding profiles between asymptomatic individuals and those who manifest disease Localization of the nucleotide sequences within promoters of interest where transcription factors bind. Identification of virus promoter binding proteins Techniques that are useful in studying functional binding of nuclear proteins to DNA sequences 1. Electrophoretic mobility shift assays (EMSA) 2. DNase I footprinting 3. Chromatin immunoprecipitation (ChIP) assay 4. Promoter pull down assays
Electrophoretic mobility shift assays (EMSA) Protein DNA complexes migrate more slowly than free DNA molecules when subjected to nondenaturing polyacrylamide or agarose gel electrophoresis. The assay is also referred to as a gel shift or gel retardation assay because the rate of DNA migration is shifted or retarded upon protein binding. PROS Ability to resolve complexes of different stoichiometry or conformation Can be used qualitatively to identify sequence specific DNA binding proteins (such as transcription factors) in crude lysates In conjunction with mutagenesis, can identify the important binding sequences within a given gene s promoter region CONS DNA protein complex does not have the complexity that is seen in vivo due to the lack of chromatin structure. DNA s insufficient length makes it difficult to measure complex interactions binding sites identified by EMSA poorly predict the presence of actual binding sites in vivo. DNase I footprinting Used to identify the region of DNA binding to transcriptional factor by assessing nucleotides resistant to the nuclease Based on the observation that when a protein binds to DNA, the DNA is protected from chemicals that would otherwise cleave it. In a typical DNA footprinting experiment, a DNA fragment with a suspected protein binding site is first isolated, and then labeled with a radioactive nucleotide or another chemical that will allow further detection. Once labeled, the DNA is then mixed in a test tube with a DNA binding protein and a chemical that cleaves the DNA, such as the enzyme DNase I. In a separate test tube, more of the same labeled DNA is mixed with the same cleaving chemical, but without the binding protein. The DNA fragments in each tube are incubated long enough for the molecule to cleave once, and then are fractionated in a DNA sequencing gel. If the DNA does contain protein binding sites, these are protected from cleavage in the test tube that contains the DNA binding protein
Chromatin immunoprecipitation assay (ChIP) spatial and temporal mapping of chromatin bound factors in vivo: 1. Whether a protein is bound 2. Where it is located 3. Whether the interaction with DNA is direct or indirect ChIP Technique 1. Crosslinking of live cells with formaldehyde (penetrates biological membranes readily, allowing the crosslinking to be done with intact cells), which reduces the risk of redistribution or reassociation of chromosomal proteins during the preparation of cellular or nuclear extracts. Chemical targets for formaldehyde are primary amino groups (lysine amino group and side chains of adenine, guanine, and cytosine) which leads to the crosslinking of both protein protein and protein DNA. Both types of crosslinks can be reversed by heating (65 C for protein DNA, boiling for protein protein). 2. Cells are lysed, and crude extracts are sonicated to shear the DNA. Short DNA fragments provide higher mapping resolution and provide the precise site on a particular chromosome of chromatin associated proteins. Extensive sonication is a way to generate fairly uniformly sized pieces of DNA. 3. Proteins and crosslinked DNA are immunoprecipitated. Protein DNA crosslinks in the IP material are then reversed, and the DNA fragments are purified. If the protein under investigation is associated with a specific genomic region in vivo, DNA fragments of this region should be further enriched in the IP compared to irrelevant portions of the genome. 4. The presence of the relevant genomic regions in the IP is determined by PCR amplification with specific primers from the region in question and reference region. A PCR product from the region in question and the reference region, obtained in the IP relative to the IP ed whole cell extract, allows quantification of the enrichment of the region of interest. CONS not useful in isolating and identifying individual family members, sensitive to weak or partial DNA binding, and the precise identity of the protecting complex cannot be elucidated
Anchored virus promoter binding assay (anchored transcriptional promoter (ATP) binding assay) Amplify promoter by PCR with an amine group linked at the 5 end The free amine group of the amplified PCR product (Amine promoter) coupled to activated Sepharose beads Pull down DNA binding proteins from cellular nuclear extracts Proteins that bound to the ATPs are run in 1D or 2D PAGE Proteins detected with antibodies in Western blot or by mass spec Variation: Use a biotinylated promoter or some other tag