Viral proteomics: The emerging cutting-edge of virus research

Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment. They encode a wide spectrum of multifunctional proteins, which interplay with and modify proteins in host cells. Viral genomes were chronologically the first to be sequenced. However, the corresponding viral proteomes, the alterations of host proteomes upon viral infection, and the dynamic nature of proteins, such as post-translational modifications, enzymatic cleavage, and activation or destruction by proteolysis, remain largely unknown. Emerging high-throughput techniques, in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes, have been applied to define viruses and their interactions with their hosts. Here, we review the major areas of viral proteomics, including virion proteomics, structural proteomics, viral protein interactomics, and changes to the host cell proteome upon viral infection.     

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.     

Polypeptides controlling hematopoietic cell development and activation. I. In vitro results

Recombinant DNA technology has been central in answering some of the most relevant questions in the research of regulation of the functional status of hematopoietic progenitor cells and their progeny. This leading article will focus on recent results that have emerged from studies utilizing recombinant molecules that control hematopoietic blood cell development and activation. The following features will be detailed: The molecular and biological characteristics and biochemistry of hematopoietic growth factors, synergizing factors and releasing factors, their role in the regulation of hematopoiesis and activation of normal and leukemic cells, their cellular sources, and regulation of production.     

Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A resolution, respectively. Strikingly, both these proteins adopt a Bcl-2-like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2-like proteins. Unlike cellular and viral Bcl-2-like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2-like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2-like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2-like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2-like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.     

病毒RNA如何逃避宿主细胞的降解

细胞RNA的降解机制不仅在基因表达调节方面具有重要作用,而且也是一种重要的病毒防御机制. 作为一种必须在细胞内增殖的微生物,病毒已经进化出了多种机制,以保护它们的RNA免被宿主细胞降解,如病毒RNA模拟宿主细胞mRNA的结构、形成磷脂包膜、形成局部二级结构、结合自己或宿主细胞编码的蛋白质和编码核酸酶增强宿主细胞mRNA降解等. 本文主要论述了病毒RNA逃避宿主细胞降解的方式,并对其应用前景进行了展望,尤其是在研发抗病毒药物方面的应用前景.     

Mast cells and basophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner

Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via FcepsilonRI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.     

Receptor tyrosine kinases and the control of hematopoiesis

The development of the hematopoietic system has served as a paradigm for addressing broad questions concerning the control of cell fate during embryogenesis, cell lineage determination, the control of gene expression during differentiation, and the molecular basis of cancer. Mature hematopoietic cells arise as the result of the developmental and proliferative decisions of a hierarchy of cells that either are developmentally pluripotent or are committed to a particular cell lineage. Recent studies have suggested that many, if not all, of these decisions are mediated through the interaction of ligands or growth factors produced by non-hematopoietic or stromal cells with transmembrane receptors expressed on hematopoietic cells. These receptors can be divided into two broad classes: receptors like the CSF-1 and Kit receptors that possess intrinsic tyrosine kinase activity and receptors without intrinsic tyrosine kinase activity but whose activation also stimulates tyrosine phosphorylation of a variety of proteins. These studies suggest a model for hematopoiesis in which the evolution of a multitude of receptor and non-receptor tyrosine kinases has allowed the emergence of a complex cellular system that utilizes variations of a single biochemical strategy to make and execute developmental decisions.     

Level of ICAM-1 Surface Expression on Virus Producer Cells Influences both the Amount of Virion-Bound Host ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity

Using virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.     

An siRNA Screen of Membrane Trafficking Genes Highlights Pathways Common to HIV-1 and M-PMV Virus Assembly and Release

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.     

Delivering cellular therapies: lessons learned from ex vivo culture and clinical applications of hematopoietic cells

Advances in stem cell biology and cellular therapy have led to promising treatments in a range of incurable diseases. However, it is unclear whether primitive stem cells can be delivered to damage tissue for regeneration of functional mature cells or stem cells must be stimulated to differentiate into mature cells in vitro and these cells delivered to patients. A range of other questions remains to be determined including how to formulate cellular products for in vivo delivery and how to undertake pharmacological testing of cellular products. Insights into these questions can be obtained from hematopoietic stem cells (HSC) which have been used for the past 50 years in bone marrow transplantation for regeneration of blood cells in patients undergoing high dose chemotherapy to treat cancer. The differentiation of HSC into mature blood cells is controlled by proteins called hematopoietic growth factors and these factors have been used to generate cellular products in vitro for clinical applications. This chapter will review some of the results of cellular therapies performed with HSC and the lessons that can be learned from these studies.     

Investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate

Many natural product-derived lectins such as the red algal lectin griffithsin (GRFT) have potent in vitro activity against viruses that display dense clusters of oligomannose N-linked glycans (NLG) on their surface envelope glycoproteins. However, since oligomannose NLG are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. For other antiviral lectins such as concanavalin A, banana lectin and cyanovirin-N (CV-N), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host T-cells. We show that GRFT, unlike CV-N, binds the surface of human epithelial and peripheral blood mononuclear cells (PBMC) through an exclusively oligosaccharide-dependent interaction. In contrast to several other antiviral lectins however, GRFT treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human PBMC, has no measureable effect on cell viability and does not significantly upregulate markers of T-cell activation. In addition, GRFT appears to retain antiviral activity once bound to the surface of PBMC. Finally, RNA microarray studies show that, while CV-N and ConA regulate expression of a multitude of cellular genes, GRFT treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. These studies indicate that GRFT has an outstanding safety profile with little evidence of induced toxicity, T-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin.     

Application of proteomics technology for analyzing the interactions between host cells and intracellular infectious agents

Host-pathogen interactions involve protein expression changes within both the host and the pathogen. An understanding of the nature of these interactions provides insight into metabolic processes and critical regulatory events of the host cell as well as into the mechanisms of pathogenesis by infectious microorganisms. Pathogen exposure induces changes in host proteins at many functional levels including cell signaling pathways, protein degradation, cytokines and growth factor production, phagocytosis, apoptosis, and cytoskeletal rearrangement. Since proteins are responsible for the cell biological functions, pathogens have evolved to manipulate the host cell proteome to achieve optimal replication. Intracellular pathogens can also change their proteome to adapt to the host cell and escape from immune surveillance, or can incorporate cellular proteins to invade other cells. Given that the interactions of intracellular infectious agents with host cells are mainly at the protein level, proteomics is the most suitable tool for investigating these interactions. Proteomics is the systematic analysis of proteins, particularly their interactions, modifications, localization and functions, that permits the study of the association between pathogens with their host cells as well as complex interactions such as the host-vector-pathogen interplay. A review on the most relevant proteomic applications used in the study of host-pathogen interactions is presented.     

SLAM and CD31: signaling molecules involved in cytokine secretion during the development of innate and adaptive immune responses

Immune cells are modulated through the crosslinking of receptors named "immunoreceptors". Ligation of immunoreceptors by their ligands induces a tyrosine-phosphorylation signal that is essential for cell activation or inhibition. Physiologically, immunoreceptor triggering is not enough for cell activation, and stimulation of co-receptors is necessary for antigen-evoked cytokine production. Thus, signal transduction pathways mediated by proteins that regulate cytokine secretion are critical to achieve an effective immune response of the host, where the balance between positive and negative signaling allows effective immune responses, preventing tolerance and autoimmunity. This review deals with recent studies based on the role of the receptor signaling lymphocytic activation molecule (SLAM), a signaling protein that modulates cytokine secretion by immune cells, and the transmembrane glycoprotein CD31, which plays multiple roles in cellular signaling events by modulating the balance between inhibitory and stimulatory signals to immune cells. Recent studies have shed light on the ability of these molecules to transmit different signals that regulate the ability of innate and adaptive immune cells to synthesize stimulatory and inhibitory cytokines.     

The dark side of circulating nucleic acids

Free circulating or cell‐free DNA (cfDNA), possibly from dying cells that release their contents into the blood as they break down, have become of major interest as a source for noninvasive diagnostics. Recent work demonstrated the uptake of human cfDNA in mouse cells in vitro and in vivo, accompanied by the activation of a cellular DNA damage response (DDR) and the appearance of apoptotic proteins in the host cells. By acting as a source of mobile genetic elements, cfDNA could be a continuous source of DNA mutagenesis of healthy cells in the body throughout life, promoting progressive cellular aging in vivo. As such, cfDNA may causally contribute to multiple aging‐related diseases, such as cancer, diabetes, and Alzheimer's disease.     

Modulation of PECAM-1 expression and alternative splicing during differentiation and activation of hematopoietic cells

PECAM-1 (CD31) is a member of immunoglobulin gene superfamily, which is highly expressed on the surface of endothelial cells and at moderate levels on hematopoietic cells. Hematopoietic cells and platelets, like endothelial cells, express multiple isoforms of PECAM-1. However, the identity and physiological role of these isoforms during hematopoiesis remains largely unknown. Here we demonstrate that PECAM-1 expression is dramatically up regulated upon phorbol myristate acetate (PMA) or transforming growth factor (TGF)-beta1-mediated differentiation of leukemic HEL and U937 cells. The level of PECAM-1 expression did not significantly change during activation of Jurkat T cells by PMA or phytohaemagglutinin (PHA). Utilizing RT-PCR and DNA sequencing analysis, we show that the expression of PECAM-1 isoforms changes in a cell-type and lineage specific manner during cellular differentiation and activation. We identified a number of novel PECAM-1 isoforms previously not detected in the endothelium. These results demonstrate that regulated expression of PECAM-1 and its exonic inclusion/exclusion occurs during differentiation and/or activation of hematopoietic cells. Thus, different PECAM-1 isoforms may play important roles in generation of hematopoietic cells and their potential interactions with vascular endothelium.     

Rap G protein signal in normal and disordered lymphohematopoiesis

Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy.