VIRAL EVASION AND MODULATION OF THE IMMUNE SYSTEM

INTRODUCTION
Many organisms mount a remarkable defense against viral infection. This response to viral infection consists of an innate/nonspecific component and an adaptive/specific defense. The innate response is considered the first line of immune defense because it is active even before infection begins. In fact, many viral infections are halted by the innate immune system, which responds very quickly within minutes to hours after infection. In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. The innate principle effector cells recognize pathogen associated molecular patterns (PAMPs) e. g viral proteins or viral RNA and DNA via a variety of pattern recognition receptors (PRPs) which include Toll like receptors, Natural killer receptors and Mannose binding receptors. These cells then release a variety of proinflammatory cytokines and chemokines which recruit inflammatory cells to the site of infection and initiate inflammation and antiviral immune response. These soluble mediators also activate macrophages, natural killer cells and dendritic cells. Most effective innate responses against viral infections are mediated by Interferon (IFN) and by activation of NK cells. IFNs increase the expression of MHC I and II molecules and of the co-stimulatory molecules on the surface of Antigen Presenting Cells (APCs).

Viral survival and ability to cause disease in their hosts depends on the ability to avoid detection and elimination by the innate and adaptive immune responses of the host Viruses have developed various strategies to subvert hosts antiviral responses to ensure their own replication and survival. Viruses have evolved elegant mechanisms to evade detection and destruction by the host immune system. Both DNA and RNA viruses have evolved similar gene functions that target many common cellular targets for immunoevasion. The mechanism of viral immune evasion and immune modulation can broadly be divided into three categories: 1

1. Impairment of the humoral immune response 2. Impairment of the cellular immune response 3. Viral interference with immune effector functions

IMPAIRMENT OF THE HUMORAL IMMUNE RESPONSE This mechanism enables the virus to avoid recognition by the humoral immune response, for example by changing its immunodominant epitopes. For example the Influenza virus-the human body can rapidly clear the virus by developing protective immunity towards it, mainly by directing neutralizing antibodies against the major surface protein of the influenza virus, hemagglutinin. Influenza virus type A has evolved two forms of antigenic variation. First, antigenic drift is caused by point mutations in the genes encoding the hemagglutinin and neuraminidase. Although the virus evades neutralization by antibodies, severe disease will not develop, since the unaltered epitopes are still being recognized. Second, in the process of antigenic shift, RNA segments are exchanged between viral strains in a secondary host, which lead to major changes in the hemagglutinin protein on the viral surface. In such cases, antibodies produced in response to previous infections cannot recognize the virus.

IMPAIRMENT OF THE CELLULAR IMMUNE RESPONSE To establish lifelong infections, viruses must achieve persistence in the face of a vigorous host immune response and this is solved by impairment of the cellular immune response in the following ways:

A. Interference with proteasomal degradation Proteasomal degradation is dependent on proteolytic cleavage of specific sequences within the protein. By altering part of its genome and thereby the viral proteins, a virus can escape processing of these proteins into peptides. Peptides that are presented in the context of MHC class I molecules result from the degradation of viral proteins by proteasomes in the cytosol. Epstein Barr Virus (EBV) is a DNA virus and it encodes a nuclear protein Epstein Barr Nuclear Antigen-1 (EBNA-1) which is essential for the replication of the viral episome in dividing virus-infected or transformed cells. EBNA-1

is a phosphoprotein that contains a 239 residue stretch of Glycine-Alanine co-repeat which inhibits its degradation by the 26S proteasome, thus reducing the pool of EBNA-1 derived peptides that could be presented with MHC I on the cell surface. The GlycineAlanine co-repeat domain is a cis-acting inhibitor of ubiquitin proteasome proteolysis. Human Cytomegalovirus (HCMV) is a DNA virus whose matrix protein pp65 results in phosphorylation of several HCMV proteins and phosphorylation of threonine residues may severely restrict access of the protein to proteasomal degradation.

B. Interference with peptide transport by transporter associated with antigen presentation (TAP) Peptides generated by proteasomal degradation in the cytosol pass the membrane of the Endoplasmic Reticulum(ER) through translocation by TAP, for assembly into ternary MHC class I complex. Many viruses can inhibit the loading of antigenic peptides onto MHC class I molecules by blocking functions of TAP. In its attempt to evade the cytotoxic T cell recognition, HCMV encodes several genes that target MHC class I molecules at different points in their assembly pathway. Expression of the HCMV-encoded unique short 6(US6) gene product results in inhibition of TAP function. US6 is a 21kDa type I transmembrane glycoprotein expressed during both early and late phases of viral infection. It inhibits the TAP function by interacting with the TAP complex on the luminal face of the ER, hence occluding the exit pore of the TAP complex without affecting peptide binding to TAP. Herpes viruses are DNA viruses. Several herpes viruses express proteins that inhibit the TAP complex. Herpes Simplex Viruses (HSV) 1 and 2 express a soluble 9KDa immediate early (IE) cytoplasmic protein ICP47, which blocks the peptide-binding site of TAP by interacting with both TAP1 and TAP2 on the cytosolic side of the ER hence competiting with the peptides. The bovine herpesvirus-1-encoded protein UL49.5 is a potent inhibitor of TAP. It inhibits TAP by inducing a conformational arrest of the transporter as well as by targeting TAP to proteasomal degradation.

C. Retention of MHC class I molecules in sub-cellular compartments

The expression of the HCMV unique short 3(US3) protein impairs maturation and intracellular transport of MHC class I heavy chains by the formation of a complex with 2 microglobulin-associated class I heavy chains prior to peptide loading in the ER. The adenoviral E3-19k gene product is translocated to ER and glycosylated. In the ER, the protein binds to MHC class I heavy chains with high affinity, inhibiting terminal glycosylation of MHC class I antigens, there by preventing their translocation to the cell surface. The murine CMV (MCMV) gene m152 encodes a 37kDa type I glycoprotein expressed early during infection the m152 gene product (gp40) retains mouse MHC I within the ERGolgi intermediate compartment (ERGIC) a process that requires the luminal part of the protein. Thus, this prevents surface expression of MHC I. The MCMV gene m06 encodes a 48kDa glycoprotein (gp48) that binds tightly through its luminal/transmembrane region to MHC class I molecules and redirects their transport to the lysosomes.

D. Destruction of MHC class I heavy chains HCMV encodes two gene products US2 and US11 that selectively target MHC I heavy chains for degradation by the proteasome. US2 binds to the MHC class I molecules during their glycosylation leading to their retrograde transport to the cytoplasm and their degradation.

E. Induction of functional paralysis of dendritic cells Immature DCs in peripheral tissues take up and process antigens, an event that triggers their differentiation into mature DCs. Mature DCs have a reduced Antigen processing capacity and an increased cell surface expression of MHC and co-stimulatory molecules. MCMV can infect immature DCs and thus induces a functional paralysis leading to reduction of expression of MHC and co-stimulatory molecules on the cell surface of infected DCs. They remain unresponsive to maturation stimuli, lose their capacity to secrete IL-12 or IL-2 and are unable to prime an effective T-cell response.

F. Down regulation of MHC class II expression

The expression of MHC class II molecules on the surface of professional APC is essential for presentation of foreign antigenic peptides to CD4 T lymphocytes. Viruses encode proteins that may interfere with expression of MHC class II antigens. Little is known about the target of viral proteins in the MHC class II peptide presentation pathway, which focuses largely on events in the endocytic pathway. The expression of HCMV protein US2 causes degradation by proteasomes of two essential proteins in the MHC class II antigen presentation pathway: the HLA-DM- and HLA-DR- chains.

Ag presentation and T-cell proliferation was found to be inhibited by undefined soluble factors, excluding IL-10 and TGF-beta1, produced by the HCMV-infected cells. In HCMV-infected cells stimulated IFN-gamma could not induce expression of MHC class II molecules, probably through the enhancement of Janus kinase1 (JAK1) protein degradation by the proteasome.

G. Circumvention of NK cell-mediated killing Down regulation of MHC class I complexes on the cell membrane by viral proteins results in elimination of the infected cells by Natural killer (NK) cells. NK cells express both activating and inhibitory surface receptors, such as CD94 and immunoglobulin-like transcript-2 (ILT-2). Herpes simplex virus (HSV) and HCMV induce NK cell cytotoxicity by down regulation of HLA-C molecules in vitro. Furthermore, HCMV avoid NK cell-mediated killing by expression of MHC homologues that engage killer cell inhibitory receptors (KIRs) and function as viral decoys to prevent NK-mediated cytotoxicity. Both HCMV and MCMV encode MHC class I heavy chain homologues: HCMV encodes gpUL18 (amino acid sequence identity with human polymorphic MHC class I molecules: 21%) MCMV encodes m144 (amino acid sequence identity with corresponding part of mouse MHC class I extra-cellular region: 25%)

H. Down regulation of transcription of proteins involved in the MHC class I peptide presentation pathway

In adenovirus 12-transformed cell lines, proteins involved in the MHC class I peptide presentation pathway, including TAP1, TAP2, LMP2, and LMP7, exhibit a >100-fold reduction in their steady state mRNA levels, with both mRNA and protein products barely detectable. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II), that is up regulated in adenovirus 12-transformed cells, have strong DNA binding activity. MHC class I transcription is down regulated through this molecule.

I. Antigenic variation of T-cell epitopes Variation in a relatively broad region can lead to alteration of peptide binding to MHC or reduction in the affinity of peptide-MHC interaction, which results in unstable peptideMHC complexes. For example, in HIV-infected individuals variants evolve in which peptide epitopes are mutated in such a way that these epitopes function as antagonists, actively silencing HIVspecific T-lymphocytes. This phenomenon is also described for amino acid changes within the core molecule of hepatitis B virus (HBV).

J. Inhibition of tapasin Adenovirus E3-19k protein has a second mechanism to affect MHC class I expression. E3-19k is capable of binding to both class I and TAP, acting as a competitive inhibitor of tapasin. This protein causes a decrease in association between these molecules and a delay in class I maturation.

K. Down regulation of CD4 expression Recognition of an antigenic determinant-containing MHC complex by T-cell receptor requires the co-expression of CD4 or CD8 molecules on the surface of T-cells. HIV-1 Vpu, Env, and Nef use different mechanisms to down-modulate CD4 at a different time points during the infection: Vpu induces degradation of CD4 by the proteasome. Nef functions early and favours internalization of CD4 molecules already present on the cell surface. It also blocks transport of MHC class I molecules to the cell surface via PI3 kinase dependent pathway.

Env forms a complex with CD4 in the Endoplasmic Reticulum

L. Down regulation of the T cell receptor Some viruses are capable of avoiding immune recognition by down regulation of the CD3/T-cell receptor (TCR) complexes on T-cell. Human herpes virus (HHV)-6A, but not HHV-6B, is able to down regulate the TCR complex at a transcriptional level, but mechanism is still unknown.

VIRAL INTERFERENCE WITH IMMUNE EFFECTOR FUNCTIONS Viral interference with cytokine synthesis and function. E.g. homologue of the immune modulator vIL-10 in the genome of HCMV. The vIL-10 has evolved to retain its inflammatory properties similar to the cellular IL-10 but has lost the immunostimulatory properties. G-Protein-coupled receptors (GCR). E.g. HCMV encodes four GCR homologues, UL33, UL78, US27, and US28. Interference with cellular signaling. E.g. HCMV infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) Inhibition of apoptosis. The cellular proteins implicated in the control of apoptosis are targeted by viral anti-apoptotic mechanisms viruses inhibit activation of caspases, encode homologs of the anti-apoptotic protein Bcl-2, block apoptotic signals triggered by activation of TNFR family members by encoding death-effector-domain-containing proteins, and inactivate IFNinduced PKR and the tumor suppressor p53, both of which promote apoptosis. Interference with complement system E.g. the gamma herpes virus and herpes virus saimiri ecode two complement control proteins;HSV vCD59 has homology with human CD59 and is able to inhibit membrane attack complex formation. Herpes virus saimiri complement control protein homologue

(CCPH) blocks C3 convertase activity and cell lysis and also reduces surface

deposition of C3. SPICE (smallpox inhibitor of complement enzyme) binds with C4b and C3b it acts as a cofactor.

PREDISPOSITION OF AN INFECTED CELL TO SUPERINFECTION Manipulation of the immune system by a virus can lead to superinfections with other viruses or bacteria that are taking advantage of the down modulated immune system. Opportunistic infections, such as HCMV and Pneumocystis carinii infections in AIDS patients, are believed to occur as a consequence of the destruction of the immune system by HIV.

REFERENCES 1. Lannello et al (2006), Viral strategies for evading antiviral cellular immune responses of the host. Journal of Leukocyte Biology. 2. Tortorella et al (2000), Viral subversion of the immune system. Annu Rev Immuno 18:861-926. 3. Hiroko Yamada (2003), Viral immune evasion.