Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.Cytomegaloviruses (CMVs) are ubiquitous pathogens causing morbidity in immune suppressed and immunodeficient hosts (34). Since CMVs are strictly species-specific viruses, the infection of mice with murine CMV (MCMV) represents a widely used model for studying CMV infection and disease (22, 40).Natural killer (NK) cells play a crucial role in the control of many viruses and are among the first cells to sense proinflammatory cytokines, as well as the perturbations in the expression of major histocompatibility complex (MHC) class I molecules and other surface molecules induced by viral infection (13). Both human CMV (HCMV) and MCMV have evolved strategies to compromise innate immunity-mediated by NK cells (20, 49).Although proinflammatory cytokines released during the early stage of MCMV infection induce NK cell activation, this is usually not sufficient for virus control (11). Namely, most mouse strains fail to mount an effector phase of NK cell response against infected cells (42), in spite of the fact that MCMV infection causes the downmodulation of MHC I molecules (17), which should activate NK cells via a “missing-self” mechanism (28). The lack of NK cell activation by MCMV is even more puzzling considering that NK cells possess activating receptors that recognize cellular ligands induced by infection. Among these is the activating receptor NKG2D, a C-type lectinlike receptor encoded by a single gene in humans and rodents (39). Engagement of NKG2D transduces a strong activating signal to promote NK cell stimulation. NKG2D also serves as a costimulatory receptor on CD8⁺ T cells (2). Several NKG2D ligands have been described in mice: MULT-1, H60a, H60b, H60c, and RAE-1α, -1β, -1γ, -1δ, and -1ɛ isoforms (4-6, 10, 14, 32, 35, 44). What prevents the activation of NK cells via the NKG2D receptor during MCMV infection? We and others have characterized four MCMV proteins involved in the downmodulation of NKG2D ligands (15, 23, 24, 26, 29, 30). Furthermore, the deletion of any of the four MCMV inhibitors of NKG2D ligands rendered virus mutants susceptible to NK cell control in vivo. The MCMV immunoevasin of NKG2D described first was the glycoprotein gp40, encoded by the gene m152 (23). Note that m152 also compromises the CD8⁺ T-cell response by downregulation of MHC class I molecules (25, 54). Later, it was noticed that m152 also affects the expression of RAE-1 proteins (29). It is important to point out that mouse strains express different RAE-1 isoforms. Some strains, such as BALB/c, express RAE-1α, -1β, and -1γ, while others, such as C57BL/6, express RAE-1δ and -1ɛ (29). All five RAE-1 isoforms are glycosylphosphatidylinositol (GPI)-linked proteins and contain MHC class I-like α1 and α2 domains (6, 10, 14, 35).Based on our initial observation that there is NKG2D-dependent control of wild type (WT) MCMV in certain mouse strains, we postulated NKG2D ligands that resist virus mediated downmodulation. We show here that the RAE-1 proteins differ in their susceptibility to downregulation by MCMV. In contrast to RAE-1γ, representing the sensitive isoform, surface-resident RAE-1δ remains present on MCMV-infected cells. The differential downmodulation of RAE-1 isoforms during MCMV infection is caused by differences in the stability of the mature RAE-1 molecules associated with a sequence motif absent in RAE-1δ.     

The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DR_L and DR_R. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DR_L, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DR_R sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.Human herpesvirus 6 (HHV-6) is a member of the Betaherpesvirus subfamily, as recently reviewed (46). The virus can enter hematopoietic cells, including T cells, B cells, natural killer (NK) cells, monocytes, and dendritic cells (DCs), as well as nonhematopoietic cells, as reviewed in references 8, 17, and 46. In culture, the virus replicates in activated peripheral blood lymphocytes (PBLs), cord blood mononuclear cells (CBMCs), and in T-cell lines (1, 17, 46). HHV-6 isolates fall into two distinct classes designated as HHV-6A and HHV-6B variants. The two variants can be distinguished by their restriction enzyme patterns, antigenicity, DNA sequences, and disease association (1, 36, 46). HHV-6B is the causative agent of roseola infantum, a prevalent children''s disease characterized by high fever and skin rash (47). In rare cases, the virus exhibits neurotropism and has been found in children experiencing convulsions up to lethal encephalitis (1, 21, 46, 48).HHV-6B reactivation from latency was found to occur in patients receiving immunosuppressive treatment in bone marrow and other transplantations. This was associated with febrile illness, delayed transplant engraftment, and neurological involvement, up to lethal encephalitis (5, 13, 34, 46). HHV-6A has thus far no clear disease association, although several studies have suggested central nervous system (CNS) tropism, including aggravation of symptoms in patients with multiple sclerosis (MS) (6, 14, 33, 41).HHV-6A and HHV-6B share general genomic architecture. The unit-length DNA molecules are approximately 160 kb, composed of a 143-kb unique (U) segment flanked by left and right direct repeats (DR_L and DR_R, respectively) (19, 24, 27, 46). The DRs are of sizes 8 to 10 kb in different viral isolates (2, 19, 24, 46). In both the HHV-6A and HHV-6B genomes, the herpesvirus conserved cleavage/packaging signals pac-1 and pac-2 (9, 15, 17) are located at the left and the right termini of the DRs (17, 19, 46). The PubMed sequence for the U1102 strain (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001664","term_id":"1344462938","term_text":"NC_001664"}}NC_001664) starts with the pac-1 signal at positions 1 to 56, followed by multiple copies of perfect and imperfect telomere-like sequences, up to position 418. It was suggested that the telomeric repeats may have originated from host cell chromosomal telomeres (43). Additionally, the DR encodes several open reading frames (ORFs), four of which are dealt with in our paper: (i) the spliced DR1 at positions 501 to 759 and 843 to 2653; (ii) DR5 at positions 3738 to 4164; (iii) the spliced DR6 at positions 4725 to 5028 and 5837 to 6720; and (iv) an ORF of DR7, at positions 5629 to 6720, partially overlapping the DR6 gene (20). Hollsberg and coworkers (37) have recently found that the homologous gene in HHV-6B encodes a nuclear protein that forms a complex with viral DNA processivity factor p41. Gompels and coworkers have also shown that DR1 and DR6 are partly homologous to the human cytomegalovirus (HCMV) US22 gene family. Both have a CXC motif: DR1 with homology to the HCMV US26 gene and DR6 with homology to the HCMV US22 gene (20). The map continues with reiterated perfect hexanucleotide telomeric sequences (GGGTAA)_n at positions 7655 to 8008 (19, 43). The number of telomeric repeats was found to vary in different viral strains (2, 43). The DR terminates with the pac-2 signal.We have recently cloned the intact HHV-6A genome into bacterial artificial chromosomes (BACs), by direct cloning of unit-length DNA produced from circular or head-to-tail replication intermediates into modified BAC vectors containing the green fluorescent protein (GFP) marker and ampicillin-puromycin (Amp-Puro) selection cassette (3). Surprisingly, the HHV-6A BAC clones as well as the parental HHV-6A (U1102) propagated in our laboratory in SupT1 cells were found to contain DRs of ∼2.7 kb instead of the expected ∼8- to 10-kb DRs, as in the early publications (19, 24, 27, 46) and in the PubMed sequence. This has raised the following questions. When did the deleted DRs arise? What was the detailed structure of deleted DRs?HHV-6 was discovered by Gallo and colleagues in 1986 (35), and viral isolates were obtained in multiple laboratories from AIDS patients, patients with lymphoproliferative disorders, and patients with roseola infantum (12, 26, 42, 45, 47). The isolates were propagated initially in activated PBLs and CBMCs and then in continuous T-cell lines, including HSB-2, J-JHAN, SupT1, Molt-3, and MT-4 (11, 45). The U1102 strain isolated by Downing and colleagues (12) was contributed to our laboratory by Robert Honess and was propagated first in activated PBLs and CBMCs (11, 18, 36, 45) and then in J-JHAN and SupT1 T cells (4, 30). To answer the question with regard to the origin of the short DRs and their structure, we have compared earlier viral HHV-6A passages with the currently propagated virus and the HHV-6A BAC clones. We describe here the detailed structure of the DR_L and DR_R of the “new” virus, containing the short ∼2.7-kb DR. We show that the deletion contained the left multiple repeats of telomere-like sequences and the ORFs from DR1 up to the DR6 first exon. Review of viral passaging since 1992 indicated that the deletion occurred spontaneously. The deleted viruses were stably and efficiently propagated in SupT1 T cells, indicating that the DR1 and DR6 first exons are not essential for virus in vitro replication.     

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.Virulent strains of Newcastle disease virus (NDV) infect domestic poultry and other birds, causing a rapidly spreading viral disease that affects the alimentary and respiratory tracts as well as the central nervous system (55). In humans, however, NDV is well tolerated (17, 18). Other than mild fever for a day, only a few adverse effects have been reported. NDV, also known as avian paramyxovirus 1, is an enveloped virus containing a negative-sense, single-stranded RNA genome which codes for six proteins in the order (from 3′ to 5′) of nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large polymerase protein (19). There are many different strains of NDV, classified as either lytic or nonlytic for different types of cells. Lytic and nonlytic NDV strains both replicate much more efficiently in human cancer cells than they do in most normal human cells (43). Viruses of both strain types have been investigated as potential anticancer agents (30, 49, 52). The NDV strains that have been evaluated most widely for the treatment of cancer are 73-T, MTH-68, and Ulster (1, 7, 11, 17, 18, 53, 54, 56, 71).Initial binding of NDV to a host cell takes place through the interaction of HN molecules in the virus coat with sialic acid-containing molecules on the cell surface (31). NDV neuraminidase has strict specificity for the hydrolysis of the NeuAc-α2,3-Gal linkage, with no hydrolysis of the NeuAc-α2,6-Gal linkage (41).NDV infection of tumor cells not only improves T-cell responses (53, 58, 68), but has also been reported to vigorously stimulate innate immune responses. In the course of NDV infection, large amounts of alpha interferon (IFN-α) are released (68) and in turn activate dendritic cells and NK cells and polarize, in concert with interleukin-12 (IL-12), toward a Th1 T-cell response (33, 44, 47). In addition, NDV induces antitumor cytotoxicity in murine macrophages which produce increased amounts of tumor necrosis factor alpha (TNF-α) and nitric oxide (51, 60) and in human monocytes through the induction of TRAIL (64). Little is known about the NDV-mediated activation of NK cells. The coincubation of peripheral blood mononuclear cells with NDV was shown previously to stimulate NK-mediated cytotoxicity (70). Enhanced cytotoxicity correlates with the induction of IFN-α (70). It is not known, however, whether NDV-infected cells can directly activate NK cells and, if so, which molecular interactions are involved.The cytolytic activity of NK cells against virus-infected or tumor cells is regulated by the engagement of activating or inhibitory NK cell surface receptors, the actions of cytokines, and cross talk with other immune cells (32, 39). Most inhibitory receptors recognize particular major histocompatibility complex (MHC) class I alleles and thereby ensure the tolerance of NK cells against self antigens (38). Activating receptors on human NK cells include CD16; NKG2D; the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46; as well as NKp80; DNAM-1; and various stimulatory coreceptors (32).NCR are important activating receptors for the antitumor and antiviral activities of NK cells (5, 32, 37). Heparan sulfate has been discussed previously as a cellular ligand for NKp46, NKp44, and NKp30 (9, 26, 27), and nuclear factor BAT3, which can be released from tumor cells under stress conditions, has been described as a cellular ligand for NKp30 (42). Ligands for NKp30 and NKp44 can be detected on the surfaces and in the intracellular compartments of several kinds of tumor cells (10). Moreover, a number of pathogen-derived NCR ligands have been reported. The hemagglutinin protein of influenza virus and the HN of Sendai virus can bind to NKp46 and NKp44 and activate NK cells (3, 24, 34). The pp65 protein of human cytomegalovirus has been shown to bind NKp30 and inhibit its function (4). Human immunodeficiency virus, vaccinia virus, and herpes simplex virus have also been shown to upregulate the expression of cellular NCR ligands in infected cells (13, 14, 62). The Plasmodium falciparum erythrocyte membrane protein 1 is involved in the NCR-mediated NK cell attack against infected erythrocytes (36). Furthermore, NKp46 recognizes cells infected with mycobacteria (22, 61), and NKp44 was recently reported to directly bind to the surfaces of mycobacteria and other bacteria (21).In this study, we investigated the expression of ligand structures for NCR and NKG2D on NDV-infected cells. We demonstrate that NDV HN proteins which are strongly expressed on NDV-infected tumor cells function as activating ligand structures for NKp44 and NKp46 but that cellular ligands for NKG2D are partially downregulated during NDV infection.     

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-Δm06m152-SIINFEKL, respectively, expressing the K^b-presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ∼10,000 K^b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ∼10% of all cell surface K^b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply.CD8 T cells recognize infected cells by interaction of their T-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77, 85, 97, 98). The number of such “peptide receptors” per cell has been estimated to be on the order of 10⁵ to 10⁶ for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9, 61, 67, 75, 76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-based epitope-specific cell sorting, as well as cells of a peptide-selected cytolytic T-lymphocyte line, protected against mCMV expressing the cognate antigenic peptide, the IE1 peptide 168-YPHFMPTNL-176 in this example, but failed to control infection with a recombinant mCMV expressing a peptide analogue in which the C-terminal MHC-I anchor residue leucine was replaced with alanine (3).Interference with the MHC-I pathway of antigen presentation has evolved as a viral immune evasion mechanism of CMVs and other viruses, mediated by virally encoded proteins that inhibit MHC-I trafficking to the cell surface (for reviews, see references 1, 24, 27, 29, 63, 70, 71, 84, and 95). These molecules are known as immunoevasins (50, 70, 89), as “viral proteins interfering with antigen presentation” (VIPRs) (95), or as negative “viral regulators of antigen presentation” (vRAPs) (34). Although the detailed molecular mechanisms differ between different CMV species in their respective hosts, the common biological outcome is the inhibition of antigen presentation. Accordingly, downmodulation of MHC-I cell surface expression is a hallmark of molecular immune evasion and actually led to the discovery of this class of molecules. Since CD8 T cells apparently protect against infection with wild-type CMV strains despite the expression of immunoevasins, the in vivo relevance of these molecules is an issue of current interest and investigation (for a review, see reference 14). As shown recently with the murine model, antigen presentation in infected host cells is not completely blocked for all epitopes, because pMHC complexes that are constitutively formed in sufficiently large amounts can exhaust the inhibitory capacity of the immunoevasins (40). Likewise, enhancing antigen processing conditionally with gamma interferon (IFN-γ) aids in peptide presentation in the presence of immunoevasins (18, 28). Thus, by raising the threshold of the amount of peptide required for presentation, immunoevasins determine whether a particular viral peptide can function as a protective epitope—an issue of relevance for rational vaccine design as well (94). Whereas deletion of immunoevasin genes gives only incremental improvement to the control of infection in immunocompetent mice (22, 51), expression of immunoevasins reduces the protective effect of adoptively transferred CD8 T cells in immunocompromised recipients (37, 40, 47, 48). In a bone marrow transplantation model, immunoevasins were recently found to contribute to enhanced and prolonged virus replication during hematopoietic reconstitution and, consequently, also to higher latent viral genome loads in the lungs and a higher incidence of virus recurrence (4). Notably, however, immunoevasins do not inhibit but, rather, enhance CD8 T-cell priming (5, 21, 22, 56), due to higher viral replication levels in draining lymph nodes associated with sustained antigen supply for the cross-priming of CD8 T cells by uninfected antigen-presenting cells (5).For mCMV, three molecules are proposed to function as vRAPs, only two of which are confirmed negative regulators that downmodulate cell surface MHC-I (34, 62, 89) and inhibit the presentation of antigenic peptides to CD8 T cells (34, 62). Immunoevasin gp40/m152 transiently interacts with MHC-I molecules and mediates their retention in a cis-Golgi compartment (96), whereas gp48/m06 stably binds to MHC-I molecules in the ER and mediates sorting of the complexes for lysosomal degradation by a mechanism that involves the cellular cargo sorting adaptor proteins AP1-A and AP3-A (73, 74). The third proposed immunoevasin of mCMV, gp34/m04 (46), also binds stably to MHC-I molecules. A function as a CD8 T-cell immunoevasin was predicted from some alleviation of immune evasion for certain epitopes and MHC-I molecules in cells infected with the deletion mutant mCMV-Δm04 (34, 42, 89), but gp34/m04 does not reduce the steady-state level of cell surface class I molecules and does not inhibit peptide presentation when expressed selectively after infection with mCMV-Δm06m152 (34, 62). The m04-MHC-I complexes are expressed on the cell surface (46) and appear to be involved in the modulation of natural killer cell activity (45).Here we give the first report on quantitating the efficacy of immunoevasins in terms of absolute numbers of pMHC complexes displayed at the cell surface. By comparing the fate of pMHC complexes already present at the cell surface in advance of immunoevasin gene expression with that of newly formed pMHC complexes, our data provide direct evidence to conclude that downmodulation of cell surface MHC-I molecules is secondary to an interruption of the flow of newly formed pMHC complexes to the cell surface.(Part of this work was presented at the 12th International CMV/Betaherpesvirus Workshop, 10 to 14 May 2009, Boston, MA.)     

Consequences of cytotoxic T-lymphocyte escape: common escape mutations in simian immunodeficiency virus are poorly recognized in naive hosts

Cytotoxic T lymphocytes (CTL) are associated with control of immunodeficiency virus infection but also select for variants that escape immune recognition. Declining frequencies of epitope-specific CTL frequencies have been correlated with viral escape in individual hosts. However, escape mutations may give rise to new epitopes that could be recognized by CTL expressing appropriate T-cell receptors and thus still be immunogenic when escape variants are passed to individuals expressing the appropriate major histocompatibility complex class I molecules. To determine whether peptide ligands that have been altered through escape can be immunogenic in new hosts, we challenged naïve, immunocompetent macaques with a molecularly cloned simian immunodeficiency virus (SIV) bearing common escape mutations in three immunodominant CTL epitopes. Responses to the altered peptides were barely detectable in fresh samples at any time after infection. Surprisingly, CTL specific for two of three escaped epitopes could be expanded by in vitro stimulation with synthetic peptides. Our results suggest that some escape variant epitopes evolving in infected individuals do not efficiently stimulate new populations of CTL, either in that individual or upon passage to new hosts. Nevertheless, escape variation may not completely abolish an epitope''s immunogenicity. Moreover, since the mutant epitope sequences did not revert to wild type during the study period, it is possible that low-frequency CTL exerted enough selective pressure to preserve epitope mutations in viruses replicating in vivo.In recent years, there has been increasing interest in AIDS vaccine approaches that elicit cytotoxic T lymphocytes (CTL), which recognize and eliminate cells infected with human immunodeficiency virus (HIV) (26). Unlike antibodies, effective CTL responses can be directed against epitopes derived from any viral protein, raising the possibility that CTL can be targeted to regions that are more conserved than the viral envelope. Current vaccine modalities can elicit potent CTL responses against multiple viral epitopes (25). Indeed, many lines of evidence indicate that cell-mediated immunity plays a major role in control of virus replication. Several studies have suggested an association between certain major histocompatibility complex (MHC) class I and class II alleles and control of viral replication or susceptibility to disease (6, 7, 11, 12, 15-17, 28, 36, 38, 39). CTL are also implicated in the initial control of immunodeficiency virus infection, since they appear in close temporal association with the reduction in peak viremia in both HIV-infected humans (5, 22) and simian immunodeficiency virus (SIV)-infected macaques (23). Antibody-mediated depletion of CD8⁺ cells in infected macaques resulted in dramatically increased virus loads in both acute and chronic infection (14, 27, 37).However, the plasticity of the viral genome also allows the generation of mutants that escape CTL recognition. Certain high-frequency CTL exert intense selective pressure on virus sequences, as revealed by the nearly total extinction of CTL-susceptible sequences from the actively replicating virus population within a few weeks of infection (2, 32). Escape from CTL has been observed in several studies of infected humans (12, 18, 21, 34, 35, 41) and macaques (2, 8, 30, 32, 40). Moreover, one report has shown that an HIV-1 escape mutant can be transmitted vertically (11), while other studies in vaccinated macaques have suggested that the evolution of escape mutants may be associated with a loss of containment of viral replication (4, 31). It therefore seems likely that escape from CTL responses occurs in most infected individuals (32).The apparent ubiquity of CTL escape may greatly complicate the design of CTL-based vaccines. The evolution of escape variants during infection of a single host may play a key part in viral persistence and therefore in the ultimate failure of immune containment and progression to AIDS. However, some investigators have suggested that T-cell receptor repertoires can recognize multiple epitope variants, so that CTL responses can coevolve along with viral escape variants in infected individuals (13). If T-cell receptor populations can recognize new variant epitopes arising within a single host, it seems plausible that variant epitope sequences could also be recognized efficiently in new hosts. Escape could also create “neo-epitopes,” novel sequences that are immunogenic to naïve T cells in individuals expressing the appropriate MHC class I molecules.The most rigorous test of the immunogenicity of epitopes altered through escape is to challenge a fully intact immune system with an escape mutant virus. Therefore, we identified common escape mutations that accumulated in immunodominant epitopes of SIVmac239 in infected macaques expressing the high-frequency MHC class I molecules Mamu-A*01 and Mamu-B*17. Together, these molecules bind three immunodominant CTL epitopes in SIVmac239: Gag_181-189CM9 (CTPYDINQM, Gag CM9) and Tat_28-35SL8 (STPESANL, Tat SL8) are bound by Mamu-A*01, and Nef_165-173IW9 (IRYPKTFGW, Nef IW9) is bound by Mamu-B*17. We have previously shown that the acute-phase response in Mamu-A*01 Mamu-B*17 double-positive macaques is dominated by CTL that recognize these three epitopes (33). We introduced common escape mutations into the SIVmac239 molecular clone and challenged macaques expressing both Mamu-A*01 and Mamu-B*17 with the mutant virus. CTL responses directed against the mutant epitopes were extremely low frequency or undetectable in fresh samples from each of the infected animals. In the absence of these responses, a completely new immunodominance hierarchy was established. Our results suggest it is unlikely that “escaped” epitopes will be recognized in newly infected individuals expressing appropriate MHC class I molecules.     

The Mitogen-Activated Protein Kinase Scaffold KSR1 Is Required for Recruitment of Extracellular Signal-Regulated Kinase to the Immunological Synapse

KSR1 is a mitogen-activated protein (MAP) kinase scaffold that enhances the activation of the MAP kinase extracellular signal-regulated kinase (ERK). The function of KSR1 in NK cell function is not known. Here we show that KSR1 is required for efficient NK-mediated cytolysis and polarization of cytolytic granules. Single-cell analysis showed that ERK is activated in an all-or-none fashion in both wild-type and KSR1-deficient cells. In the absence of KSR1, however, the efficiency of ERK activation is attenuated. Imaging studies showed that KSR1 is recruited to the immunological synapse during T-cell activation and that membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.Kinase suppressor of Ras was originally identified in Drosophila melanogaster (53) and Caenorhabditis elegans (19, 32, 52) as a positive regulator of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. It is thought to function as a MAP kinase scaffold because it can bind to Raf, MEK, and ERK (18, 19, 27, 28, 44, 59). While the exact function of KSR is unknown, preassembling the three components of the ERK MAP kinase cascade could function to enhance the efficiency of ERK activation, potentially regulate the subcellular location of ERK activation, and promote access to specific subcellular substrates (16, 45, 46).While only one isoform of KSR is expressed in Drosophila (53), two KSR isoforms have been identified in C. elegans (19, 32, 52) and most higher organisms. They are referred to as KSR1 and KSR2 (32, 43). While KSR1 mRNA and protein are detectable in a wide variety of cells and tissues, including brain, thymus, and muscle (10, 11, 29), little is known about the expression pattern of KSR2.We previously reported the phenotype of KSR1-deficient mice (30). These mice are born at Mendelian ratios and develop without any obvious defects. Using gel filtration, we showed that KSR1 promotes the formation of large signaling complexes containing KSR1, Raf, MEK, and ERK (30). Using both primary T cells stimulated with antibodies to the T-cell receptor as well as fibroblasts stimulated with growth factors, we showed that KSR1-deficient cells exhibit an attenuation of ERK activation with defects in cell proliferation.Here we explored the role of KSR1 in NK cell-mediated cytolysis. The killing of a target cell by a cytolytic T cell or NK cell is a complicated process that involves cell polarization with microtubule-dependent movement of cytolytic granules to an area that is proximal to the contact surface or immunological synapse (7, 33, 34, 48-50, 54). A variety of different signaling molecules are also involved, including calcium (23), phosphatidylinositol-3,4,5-triphosphate (13, 17), and activation of the ERK MAP kinase (6, 42, 56). Recently, the recruitment of activated ERK to the immunological synapse (IS) has been shown to be a feature of successful killing of a target by cytotoxic T lymphocytes (58).How active ERK is recruited to the synapse is not known. Since KSR1 is known to be recruited to the plasma membrane by Ras activation (24), and since the immunological synapse is one of the major sites of Ras activation (26, 41), it seemed plausible to test the hypothesis that KSR1 recruitment to the plasma membrane functions to recruit ERK to the immunological synapse and facilitate its activation. We found that KSR1 was recruited to the immunological synapse and that KSR1 appeared to be required for the localization of active ERK at the contact site. As KSR1-deficient cells exhibit a defect in killing, this suggests that KSR1 recruitment to the synapse may be important in the cytolytic killing of target cells.     

HLA Class I Subtype-Dependent Expansion of KIR3DS1+ and KIR3DL1+ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection

NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of human immunodeficiency virus type 1 (HIV-1) infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. Here we demonstrate that NK cells expressing the activating receptor KIR3DS1⁺ and, to a lesser extent, the inhibitory receptor KIR3DL1⁺ specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. These data demonstrate for the first time the HLA class I subtype-dependent expansion of specific KIR⁺ NK cells during an acute viral infection in humans.NK cells are cytotoxic effector cells that play a vital role in the innate immune response to viral infections (9, 12, 33). The critical role of NK cells in acute viral infections has been best characterized in acute murine cytomegalovirus (MCMV) infection (14, 28). While several murine lab strains are resistant to MCMV infection, others are highly susceptible. Resistance to MCMV infection was mapped to a gene encoding an activating NK cell receptor, Ly49H, which has been shown to be critical in the early recognition and control of MCMV infection via the direct recognition of a viral product (M157) expressed on infected cells (28). Remarkably, MCMV-infected mice exhibit a dramatic expansion of NK cells during acute infection, but this expansion is restricted to the specific accumulation of Ly49H⁺ NK cells (16). Data from these studies suggest that the antiviral activity of the Ly49H⁺ NK cells is linked to their ability to expand early in infection, prior to the development of adaptive antiviral immunity.While the critical role of Ly49H⁺ NK cells in MCMV infection has been well established, very little is known about the clonal composition of NK cells that expand in human viral infections, and the NK cell receptors that mediate their antiviral activity. Unlike T cells and B cells, the specificity of NK cells is not determined by a single NK cell receptor (8); rather, NK cells express an array of activating and inhibitory receptors that regulate their activity. While the expression of these receptors is stochastic, the random combinations of different receptors on the surface of a given NK cell clone determine its ability to respond to a specific target cell (26, 27). It has been suggested that individual NK cell populations expressing a specific array of receptors may respond differentially to diverse viral infections (7). This has been further supported by epidemiological studies associating the expression of individual activating or inhibitory NK cell receptors in combination with their HLA class I ligands with better or worse disease outcomes in viral infections such as hepatitis C virus (22), human immunodeficiency virus (HIV) (29, 30), human papillomavirus (11), and CMV (7). The functional basis for this protective immunity mediated by NK cells in human viral infections remains largely unknown.Similar to MCMV infection, highly functional NK cells expand rapidly in acute HIV-1 infection, prior to the induction of adaptive immune responses (2). One particular activating killer immunoglobulin-like NK cell receptor (KIR3DS1), in combination with its putative ligand, an HLA-B allele with isoleucine at position 80 (HLA-B Bw480I), has been shown to be associated with slower HIV-1 disease progression (29). We have recently shown that KIR3DS1⁺ NK cells can effectively suppress HIV-1 replication in HLA-B Bw480I⁺ target cells in vitro (1). Furthermore, a subset of inhibitory alleles from the same locus, KIR3DL1, that show high cell surface expression levels have similarly been associated with slower disease progression toward AIDS in the presence of their ligand, HLA-B Bw480I (30). These data suggest that both KIR3DS1⁺ and KIR3DL1⁺ NK cells may play a critical role in the control of natural HIV-1 infection, depending on the interaction with their ligand on infected cells (4). However, the mechanisms underlying their protective role are not understood.Given the critical role of NK cells in acute viral infections and the described expansion of NK cells overall during acute HIV-1 infection (16), we assessed clonal NK cell expansions during acute HIV-1 infection by quantitative PCR and flow cytometric analysis. Here we report an HLA class I subtype-dependent specific expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells during acute HIV-1 infection. These data demonstrate for the first time the impact of the HLA class I ligands on clonal NK cell expansions during an acute human viral infection.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.Type I interferons (IFNs), namely, alpha interferon (IFN-α) and IFN-β, are not only essential for host innate defense against viral pathogens but also critically modulate the development of virus-specific adaptive immune responses (6, 8, 28, 30, 36, 50, 61). The importance of type I IFNs in host defense has been demonstrated by studying mice deficient in the type I IFN receptor, which are highly susceptible to most viral pathogens (2, 47, 62).Recent studies have suggested that the production of type I IFNs is controlled by different innate pattern recognition receptors (PRRs) (19, 32, 55, 60). There are three major classes of PRRs, including Toll-like receptors (TLRs) (3, 40), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (25, 48, 51), and nucleotide oligomerization domain (NOD)-like receptors (9, 22). TLRs are a group of transmembrane proteins expressed on either cell surfaces or endosomal compartments. RLRs localize in the cytosol. Both TLRs and RLRs are involved in detecting viral pathogens and controlling the production of type I IFNs (52, 60). In particular, the endosome-localized TLRs (TLR3, TLR7/8, and TLR9) play important roles in detecting virus-derived double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA-containing unmethylated CpG motifs, respectively. In contrast, RIG-I detects virus-derived ssRNA with 5′-triphosphates (5′-PPPs) or short dsRNA (<1 kb), whereas melanoma differentiation-associated gene 5 (MDA5) is responsible for recognizing virus-derived long dsRNA as well as a synthetic mimic of viral dsRNA poly(I):poly(C) [poly(I·C)] (24, 60). Recognition of viral pathogen-associated molecular patterns (PAMPs) ultimately leads to the activation and nuclear translocation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), which, in turn, switches on a cascade of genes controlling the production of both type I IFNs and other proinflammatory cytokines (10, 11, 60).Lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse, is an excellent system to study the impact of virus-host interactions on viral pathogenesis and to address important issues related to human viral diseases (1, 45, 49, 67). LCMV infection induces type I IFNs as well as other proinflammatory chemokines and cytokines (6, 41). Our previous studies have demonstrated that TLR2, TLR6, and CD14 are involved in LCMV-induced proinflammatory chemokines and cytokines (66). The mechanism by which LCMV induces type I IFN responses, however, has not been clearly defined (7, 8, 31, 44). The role of the helicase family members RIG-I and MDA5 in virus-induced type I IFN responses has been recently established. RIG-I has been found to be critical in controlling the production of type I IFN in response to a number of RNA viruses, including influenza virus, rabies virus, Hantaan virus, vesicular stomatitis virus (VSV), Sendai virus (SeV), etc. In contrast, MDA5 is required for responses to picornaviruses (15, 25, 63).In the present study, we demonstrated that LCMV genomic RNA strongly activates type I IFNs through a RIG-I/MDA5-dependent signaling pathway. Our present study further demonstrated that the LCMV nucleoprotein (NP) blocks LCMV RNA- and other viral ligand-induced type I IFN responses.     

Soluble HLA-G Inhibits Myeloid Dendritic Cell Function in HIV-1 Infection by Interacting with Leukocyte Immunoglobulin-Like Receptor B2

Dendritic cells represent a specialized class of professional antigen-presenting cells that are responsible for priming and maintaining antigen-specific effector cell responses and regulating immune activation by cytokine secretion. In HIV-1 infection, myeloid dendritic cells are highly dysfunctional, but mechanisms contributing to their functional alterations are not well defined. Here, we show that soluble molecules of the nonclassical major histocompatibility complex class Ib (MHC-Ib) antigen HLA-G are highly upregulated in the plasma during progressive HIV-1 infection, while levels of membrane-bound HLA-G surface expression on dendritic cells, monocytes, and T cells only slightly differ among HIV-1 progressors, HIV-1 elite controllers, and HIV-1-negative persons. These elevated levels of soluble HLA-G in progressive HIV-1 infection likely result from increased secretion of intracellularly stored HLA-G molecules in monocytes and dendritic cells and contribute to a functional disarray of dendritic cells by inhibiting their antigen-presenting properties, while simultaneously enhancing their secretion of proinflammatory cytokines. Interestingly, we observed that these immunoregulatory effects of soluble HLA-G were mainly mediated by interactions with the myelomonocytic HLA class I receptor leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4), while binding of soluble HLA-G to its alternative high-affinity receptor, LILRB1 (ILT2), appeared to be less relevant for its immunomodulatory functions on dendritic cells. Overall, these results demonstrate a critical role for soluble HLA-G in modulating the functional characteristics of professional antigen-presenting cells in progressive HIV-1 infection and suggest that soluble HLA-G might represent a possible target for immunotherapeutic interventions in HIV-1-infected persons.The hallmark of HIV-1-associated immune deficiency is a progressive decline of T-cell immunity; however, HIV-1 infection also involves dysfunction of multiple other components of the innate and adaptive immune systems, including B cells (25, 28), NK cells (22), and NK T (NKT) cells (30). Perhaps most importantly, HIV-1 infection leads to functional deficiencies of myeloid dendritic cells (mDC) (2, 8, 10), which as professional antigen-presenting cells have critical roles in priming and maintaining adaptive and innate effector cell responses and in regulating immune activation (4). In progressive HIV-1 infection, myeloid dendritic cells show an activated phenotype, with upregulation of costimulatory molecules and maturation markers (2, 6), but their functional antigen-presenting properties are poor (7), which may be responsible for the dysfunctional properties of antigen-specific T- and B-cell responses during HIV-1 infection. In addition, mDC in progressive HIV-1 infection seem to secrete higher levels of proinflammatory cytokines (2) and by this mechanism may contribute to generalized activation and exhaustion of the immune system, two events that play important roles in the pathogenesis of HIV-1 infection (9). The molecular pathways that contribute to dendritic cell dysfunction in HIV-1 infection, however, are unclear, but their understanding holds promise for a targeted manipulation of dendritic cells for immunotherapeutic interventions.HLA-G represents a nonclassical major histocompatibility complex class Ib (MHC-Ib) antigen, which, in comparison to classical HLA class I molecules, has limited functions for antigen presentation and restriction of T-cell immune responses but important immunoregulatory properties during various infectious, inflammatory, and malignant diseases (5). Unlike expression of classical HLA class I molecules, expression of HLA-G is mostly limited to fetal trophoblastic tissues (15), but ectopic expression of HLA-G on T cells (11), monocytes, and dendritic cells (3) has been documented in a variety of pathological conditions, including HIV-1 infection (16, 19). Moreover, it is well recognized that alternative splicing of HLA-G can lead to soluble isoforms which cause systemic immunoregulatory effects in the absence of localized tissue expression. The highest-affinity receptors for HLA-G include leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2) and LILRB2 (ILT4), two members of the LILR family, as well as the NK cell receptor KIR2DL4. By interacting with such receptors, HLA-G can induce a variety of immunomodulatory effects, including inhibition of antigen-specific T-cell (17) and NK cell responses (27). How HLA-G changes the functional profile of dendritic cells during chronic viral diseases such as HIV-1 infection remains unknown.In the present study, we analyzed immunomodulatory effects of HLA-G in individuals with different rates of HIV-1 disease progression. Our studies show that soluble HLA-G in the plasma, but not membrane-bound HLA-G expression on leukocytes, is strikingly upregulated during progressive HIV-1 infection. This soluble HLA-G critically contributes to the functional deficiencies of myeloid dendritic cells by interacting with ILT4 (LILRB2), while interactions with its other high-affinity receptor, ILT2, seem to be less relevant. Overall, these data show that binding interactions between ILT4 and soluble HLA-G play a key role in mediating dendritic cell dysfunction in progressive HIV-1 infection and might represent a possible target for immunotherapeutic interventions in HIV-1 infection.     

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus''s success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.Influenza is an acute respiratory virus infection that continues to pose endemic, zoonotic, and pandemic threats to human health, with significant morbidity and mortality (17). At the early phase of viral infection, innate immunity plays important roles in host defense by limiting viral replication and helping to initiate an adaptive immune response. Natural killer (NK) cells are key effector cells in innate immunity and play a critical role in the first line of host defense against acute viral infections by directly destroying infected cells without the need for prior antigen stimulation (7, 20). As influenza illness and virus transmission usually occur in the first few days of infection, the virus has to devise strategies to evade host innate immune responses, including NK cell immunity (15, 21).NK cells can recognize and kill influenza virus-infected cells (2, 10, 23); to counteract this killing, however, influenza virus has developed an escape strategy that inhibits NK cell cytotoxicity by increasing the binding of two inhibitory receptors to the infected cells after infection (1). The individuals with complete NK cell deficiency developed life-threatening varicella zoster virus and cytomegalovirus infection, but no severe influenza virus infection occurred (30, 40). Indeed, the interaction between human NK cells and influenza virus remains poorly understood. After influenza virus infection, respiratory epithelial cells release inflammatory chemokines that recruit NK cells to the site of infection (12). As a lytic virus, numerous influenza virus particles are released from the infected epithelia and macrophages (5, 9, 33). In the infected microenvironment, NK cells undoubtedly encounter these infective virus particles. It is therefore important to investigate the direct interaction of NK cells with influenza virus. Patients with severe influenza virus infection were shown to have diminished NK cells in peripheral blood and an almost complete absence of pulmonary NK cells, together with marked apoptosis (13, 42). During influenza virus infection in mice, a transient increase of NK cytotoxicity is followed by a marked decrease in NK cell activity, with a virus dose-dependent effect (8, 28). These data suggest that influenza virus may directly target NK cells as part of its immunoevasion strategies. However, no reports of the direct effects of influenza virus on human NK cells have so far been available.In this study, we demonstrated that influenza virus infects and replicates in primary human NK cells. Viral infection was dependent on sialic acids on the cells. The entry was mediated by both clathrin- and caveolin-dependent endocytosis rather than macropinocytosis. Influenza virus infection induced a marked apoptosis of NK cells, which contributed to reduced NK cell cytotoxicity. This, to the best of our knowledge, is the first paper to demonstrate that influenza virus can directly infect NK cells and induce cell apoptosis. These findings suggest that influenza virus may have developed a novel strategy to evade NK cell innate immune defenses, which is likely to facilitate viral transmission and may also contribute to virus pathogenesis.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.