Natural killer (NK) cells are populations of lymphocytes that can be activated to mediate significant levels of cytotoxic activity and produce high levels of certain cytokines and chemokines. NK cells respond to and are important in defense against a number of different infectious agents. The first indications for this function came from the observations that virus-induced interferons alpha/beta (IFN-alpha and -beta) are potent inducers of NK cell-mediated cytotoxicity, and that NK cells are important contributors to innate defense against viral infections. In addition to IFN-alpha/beta, a wide range of other innate cytokines can mediate biological functions regulating the NK cell responses of cytotoxicity, proliferation, and gamma interferon (IFN-gamma) production. Certain, but not all, viral infections induce interleukin 12 (IL-12) to elicit NK cell IFN-gamma production and antiviral mechanisms. However, high levels of IFN-alpha/beta appear to be unique and/or uniquely dominant in the context of viral infections and act to regulate other innate responses, including induction of NK cell proliferation in vivo and overall negative regulation of IL-12 production. A detailed picture is developing of particular innate cytokines activating NK cell responses and their consorted effects in providing unique endogenous milieus promoting downstream adaptive responses, most beneficial in defense against viral infections.

NATURAL KILLER CELLS IN ANTIVIRAL DEFENSE: Function and Regulation by Innate Cytokines

Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.

Biochemical, cellular, and pharmacological aspects of the multidrug transporter

Prions cause transmissible and genetic neurodegenerative diseases, including scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Straussler-Scheinker diseases of humans. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein, which is encoded by a chromosomal gene. A posttranslational process, as yet unidentified, converts the cellular prion protein into an abnormal isoform. Scrapie incubation times, neuropathology, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neuro-degeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Understanding prion diseases may advance investigations of other neurodegenerative disorders and of the processes by which neurons differentiate, function for decades, and then grow senescent.

https://science.sciencemag.org/content/sci/252/5012/1515.full.pdf

Molecular biology of prion diseases

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Biochemical, cellular, and pharmacological aspects of the multidrug

THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Many viruses have evolved mechanisms to avoid detection by the host immune system. Herpes simplex virus (HSV) expresses an immediate early protein, ICP47, which blocks presentation of viral peptides to MHC class I-restricted cells. The properties of the newly synthesized class I molecules in HSV-infected cells resemble those of cell lines deficient in the transporter associated with antigen processing (TAP) in that class I molecules are retained in the endoplasmic reticulum, and the heavy chain and beta 2-microglobulin subunits dissociate in detergent extracts but the complex can be stabilized by peptides. We show here that ICP47 binds to TAP and prevents peptide translocation into the endoplasmic reticulum.

Herpes simplex virus turns off the TAP to evade host immunity

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.

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Brefeldin A Redistributes Resident and Itinerant Golgi Proteins to the Endoplasmic Reticulum

Cells infected with viruses or undergoing oncogenic transformation express new or altered self-proteins that may trigger host immune responses. CD8+ cytotoxic T lymphocytes (CTL) recognize such proteins in the form of small antigenic peptide fragments complexed with cell surface major histocompatibility complex (MHC) class I molecules (32, 86). Peptides presented by MHC class I complexes are generated largely from endogenously synthesized proteins through proteolysis in the cytosol, often by the action of proteasomes (22, 47, 63, 80), and are delivered to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) (49, 83, 87), where they are assembled with nascent class I molecules. The resulting complex is then transported via the secretory pathway to the cell surface (17, 55, 81, 85). Multiple factors contribute to the ability of a given determinant in a protein to elicit a CTL response. These include the efficiency of production of the peptide and transport to the ER (7, 8, 20, 26, 54, 57), peptide affinity for class I molecules (15, 82), and the frequency of potentially reactive T cells in the repertoire (12, 18, 52, 56).

We have used simian virus 40 (SV40) large tumor, or T, antigen (SV40 Tag) to understand factors which govern the immunogenicity of CTL determinants within a tumor antigen. SV40 Tag is a multifunctional 94-kDa nuclear oncoprotein which can initiate and maintain transformation of a wide variety of cell types in vitro (28). In vivo, the Tag can induce neoplasia with a metastatic potential when expressed as a transgene under control of a tissue-specific promoter or when expressed from the viral promoter (10, 34, 37, 59). In the immunocompetent host, the Tag often induces a vigorous cellular immune response which leads to the rejection of transplanted Tag-induced tumors (reviewed in references 76 and 77). Although SV40 Tag has long served as a model tumor inducer and immunogen in experimental systems, more recent detection of SV40 virus in isolates derived from human tumors has prompted renewed interest in the potential role of the immune system in controlling tumor induction by SV40 (9, 13, 41, 46).

SV40 Tag contains four H-2b-restricted CTL determinants, of which three are H-2Db restricted (19, 43, 51, 77). These include CTL determinants I (residues 206 to 215), II/III (residues 223 to 231), and determinant V (residues 489 to 497). Determinant V was previously characterized as immunorecessive within the context of the full-length Tag (50, 69). That is, determinant V-specific CTL responses were detected only (and then with some difficulty) following immunization of mice with transformed cells expressing a mutated Tag in which the three immunodominant determinants (determinants I, II/III, and IV) were deleted or rendered nonantigenic (50, 69).

To further characterize the immunological potential of the H-2b-restricted SV40 Tag CTL epitopes, including the immunorecessive determinant V, we have extended these findings by examining the antigenicity and immunogenicity of SV40 Tag CTL determinants expressed in various polypeptide contexts from recombinant vaccinia viruses (rVVs). Our results show that the immunogenicity of the immunorecessive epitope V is significantly enhanced by targeting it to the ER.

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An Endoplasmic Reticulum-Targeting Signal Sequence Enhances the Immunogenicity of an Immunorecessive Simian Virus 40 Large T Antigen Cytotoxic T-Lymphocyte Epitope

We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

/pdf/introduction-of-a-glycosylation-site-into-a-secreted-protein-tnv7db9s1m.pdf

Introduction of a Glycosylation Site into a Secreted Protein Provides Evidence for an Alternative Antigen Processing Pathway: Transport of Precursors of Major Histocompatability Complex Class I–Restricted Peptides from the Endoplasmic Reticulum to the Cytosol

The 4H84 monoclonal antibody detecting β2m free nonclassical HLA-G molecules also binds to free heavy chains of classical HLA class I antigens present on activated lymphocytes

Gustav Russ

Papers

Herpes simplex virus turns off the TAP to evade host immunity

Brefeldin A Redistributes Resident and Itinerant Golgi Proteins to the Endoplasmic Reticulum

An Endoplasmic Reticulum-Targeting Signal Sequence Enhances the Immunogenicity of an Immunorecessive Simian Virus 40 Large T Antigen Cytotoxic T-Lymphocyte Epitope

Introduction of a Glycosylation Site into a Secreted Protein Provides Evidence for an Alternative Antigen Processing Pathway: Transport of Precursors of Major Histocompatability Complex Class I–Restricted Peptides from the Endoplasmic Reticulum to the Cytosol

The 4H84 monoclonal antibody detecting β2m free nonclassical HLA-G molecules also binds to free heavy chains of classical HLA class I antigens present on activated lymphocytes