Genetic basis of viral persistence: single amino acid change in the viral glycoprotein affects ability of lymphocytic choriomeningitis virus to persist in adult mice.

This study has identified a single amino acid change in the viral glycoprotein that profoundly affects the ability of lymphocytic choriomeningitis virus (LCMV) to persist in its natural host. Adult immunocompetent mice infected with a variant of the Armstrong strain, spleen isolate clone 13 (svA/svA), harbor virus for several months and exhibit suppressed T cell responses. In contrast, adult mice infected with a reassortant virus (svA/wtA) that contains the L segment of the spleen variant and the S segment of the parental wt Armstrong, make potent LCMV-specific CTL responses and clear the infection within 2-4 wk. These two viruses, spleen variant clone 13 and the reassortant svA/wtA, are identical in their noncoding regions and show no amino acid changes in any of their viral genes except for one substitution in the glycoprotein. The reassortant virus svA/wtA has a phenylalanine at amino acid residue 260 of the glycoprotein, whereas the spleen variant clone 13 has a leucine at this position. This study constitutes one of the first reports defining the genetic basis of viral persistence at the whole animal level, and identifying a single mutation that markedly increases the ability of a virus to persist in its natural host.

Cell-mediated cytotoxicity in perforin-less mice.

We have used a perforin-less (PO) mouse to explore alternate CTL-mediated lytic pathways. PO mice are unable to overcome an infection with LCMV in vivo. Nevertheless, splenocytes from infected mice show vigorous, antigen-specific cytotoxicity that requires the presence of the Fas antigen on target cells. The Fas lytic pathway is virtually indistinguishable, in terms of kinetics and magnitude of cytotoxicity, from perforin/granzyme-mediated lysis. It is rapidly induced in CTL upon occupation of the TcR, and requires protein synthesis for full expression. Upon removal of the activating signal, the capacity for fas-mediated lysis rapidly disappears. PO mice infected with LCMV also undergo what appears to be a CD8-mediated immunopathology, and rarely live beyond one month. The precise basis of this pathology is unknown at present. Given the widespread distribution of Fas in mice, particularly on inflamed tissues, the complete failure to clear virus from any tissue or organ is surprising.

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.

Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus.

This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K-->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-->K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K-->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F-->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.

Bone marrow is a major site of long-term antibody production after acute viral infection.

Antiviral antibody production is often sustained for long periods after resolution of an acute viral infection. Despite extensive documentation of this phenomenon, the mechanisms involved in maintaining long-term antibody production remain poorly defined. As a first step towards understanding the nature of long-term humoral immunity, we examined the anatomical location of antibody-producing cells during acute viral infection. Using the lymphocytic choriomeningitis virus (LCMV) model, we found that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appears in the bone marrow and constitutes the major source of long-term antibody production. Following infection of adult mice, LCMV-specific antibody-secreting cells (ASC) peaked in the spleen at 8 days postinfection but were undetectable in the bone marrow at that time. The infection was essentially cleared by 15 days, and the ASC numbers in the spleen rapidly declined while an increasing population of LCMV-specific ASC began to appear in the bone marrow. Compared with the peak response at 8 days postinfection, time points from 30 days to more than 1 year later demonstrated greater-than-10-fold reductions in splenic ASC. In contrast, LCMV-specific plasma cell numbers in the bone marrow remained high and correlated with the high levels of antiviral serum antibody. The presence of LCMV-specific plasma cells in the bone marrow was not due to persistent infection at this site, since the virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and PCR assays. The immunoglobulin G subclass profile of antibody-secreting cells derived from bone marrow and the spleen correlated with the immunoglobulin G subclass distribution of LCMV-specific antibody in the serum. Upon rechallenge with LCMV, the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. Bone marrow ASC populations and LCMV-specific antibody levels in the serum did not change during the early phase of the reinfection, but both increased about two-fold by 15 days postchallenge. After both primary and secondary viral infections, LCMV-specific plasma cells were maintained in the bone marrow, showing that the bone marrow is a major site of long-term antibody production after acute viral infection. These results documenting long-term persistence of plasma cells in the bone marrow suggest a reexamination of our current notions regarding the half-life of plasma cells.

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.

A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo.

We describe a protein with the hallmarks of a chemokine, designated CXCL16, that is made by dendritic cells (DCs) in lymphoid organ T cell zones and by cells in the splenic red pulp. CXCL16 contains a transmembrane domain and both membrane-bound and soluble forms are produced. Naïve CD8 T cells, natural killer T cells and a subset of memory CD4 T cells bind CXCL16, and activated T cells migrated chemotactically to the soluble chemokine. By expression cloning, Bonzo (also known as STRL33 and TYMSTR) was identified as a CXCL16 receptor. CXCL16 may function in promoting interactions between DCs and CD8 T cells and in guiding T cell movements in the splenic red pulp. CXCL16 was also found in the thymic medulla and in some nonlymphoid tissues, indicating roles in thymocyte development and effector T cell trafficking.

Genetic analysis of in vivo-selected viral variants causing chronic infection: importance of mutation in the L RNA segment of lymphocytic choriomeningitis virus.

Viral variants with different biological properties are present in the central nervous systems (CNS) and lymphoid tissues of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Viral isolates from the CNS are similar to the original Armstrong LCMV strain and induce potent virus-specific T-cell responses in adult mice, and the infection is rapidly cleared. In contrast, LCMV isolates derived from spleens of carrier mice cause persistent infections in adult mice. This chronic infection is associated with low levels of antiviral T-cell responses. In this study, we genetically characterized two independently derived spleen variants by making recombinants (reassortants) between the spleen isolates and wild-type (wt) LCMV and showed that the ability to persist in adult mice and the associated suppression of T-cell responses segregates with the large (L) RNA segment. In addition, we analyzed a revertant (isolated from the CNS) derived from one of the spleen variants. By comparing the biological properties of three reassortants that contained the same S segment but had the L segment of either the original wt Armstrong LCMV, the spleen variant derived from it, or the CNS revertant derived from the spleen variant, we were able to show unequivocally that biologically relevant mutations occurred in the L segment not only during generation of the spleen variant from wt LCMV but also in reversion of the spleen variant to the wt phenotype. Thus, our results showed that (i) genetic alterations in the L genomic segment were involved in organ-specific selection of viral variants, and (ii) these mutations profoundly affected the ability of LCMV to cause chronic infections in adult mice.

Distinct CD8 T cell functions mediate susceptibility to histoplasmosis during chronic viral infection.

It has long been recognized that some viral infections result in generalized immune suppression. In acute infections, this period of suppressed immunity is relatively short. However, chronic infections associated with a prolonged period of immune suppression present far greater risks. Here, we examined the role of CD8 T cell responses following viral infection in immunity to systemic histoplasmosis. Although wild-type mice with systemic histoplasmosis were able to control the infection, those simultaneously infected with lymphocytic choriomeningitis virus clone 13 showed reduced immunity with greater fungal burden and high mortality. The immune suppression was associated with loss of CD4 T cells and B cells, generalized splenic atrophy, and inability to mount a granulomatous response. Removing the anti-viral CD8 T cells in the coinfected mice enabled them to reduce the fungal burden and survive the infection. Their lymphoid organs were replenished with CD4 T and B cells. In contrast to wild-type mice, perforin-deficient mice infected with lymphocytic choriomeningitis virus clone 13 and Histoplasma showed an absence of immunopathology, but the animals still died. These results show that CD8 T cells can suppress immunity through different mechanisms; although immunopathology is perforin-dependent, lethality is perforin-independent.

Antiviral cytotoxic T-cell memory by vaccination with recombinant Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterium that is able to escape phagocytic vesicles and replicate in the cytoplasm of infected cells. As with viral vectors, this intracytoplasmic life cycle provides a means for introducing foreign proteins into the major histocompatibility complex class I pathway of antigen presentation. Using recombinant L. monocytogenes (rLM) strains expressing the full-length nucleoprotein (NP) or a single cytotoxic T-lymphocyte (CTL) epitope from lymphocytic choriomeningitis virus (LCMV), we analyzed antiviral CTL responses induced by rLM vaccination. After vaccination, rLM was cleared from the host within 7 days while inducing an LCMV-specific ex vivo CD8+ effector CTL response. Virus-specific CTL memory was maintained for 6 months postvaccination, as demonstrated by vigorous secondary CTL responses after in vitro stimulation. A single immunization with rLM that expressed either the full-length NP gene or the CTL epitope alone resulted in LCMV NP-specific CTL precursor frequencies of approximately 1/10(4) CD8+ T cells. A second rLM vaccination resulted in enhanced virus-specific CTL activity and in vitro proliferation. rLM-vaccinated mice were protected against chronic viral infection by an accelerated virus-specific memory CTL response. These mice cleared infectious virus as well as viral antigen, suggesting that sterilizing immunity was achieved. In contrast to mice that received wild-type LM, rLM-vaccinated mice were protected from virally induced immunosuppression and splenic atrophy associated with chronic LCMV infection. The ability to elicit long-term cell-mediated immunity is fundamental in designing vaccines against intracellular pathogens, and these results demonstrate the efficacy of recombinant LM vaccination for inducing protective antiviral CTL memory.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

A role for perforin in downregulating T-cell responses during chronic viral infection.

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf -/-) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf -/- mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral "exhaustion" of activated CD8 T cells occurred less efficiently in perf -/- mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf -/- mice; approximately 50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.

Immune function in mice lacking the perforin gene.

Mice lacking the perforin gene were generated by using targeted gene disruption in embryonal stem cells. When infected with lymphocytic choriomeningitis virus (LCMV), perforin-less (-/-) mice showed clear signs of having mounted an immune response based on activation of CD8 T cells but were unable to clear the LCMV infection. This failure to eliminate virus was accompanied by a failure to generate spleen cells capable of lysing LCMV-infected fibroblasts in vitro. Spleen cells from LCMV-infected -/- mice were able to lyse hematopoietic target cells after exposure to phorbol 12-myristate 13-acetate and ionomycin, provided the target cells expressed the Fas antigen. Spleen cells from -/- mice also responded to alloantigen in mixed leukocyte culture by blastogenesis and proliferation. The resulting cells were able to lyse hematopoietic target cells, although not as well as spleen cells from +/+ littermates sensitized in the same manner. However, lysis by -/- cells was again seen only if the target cells expressed Fas antigen. We conclude that perforin-less -/- mice retain and express the Fas lytic pathway as expressed in vitro but that this pathway is insufficient to clear an LCMV infection in vivo.

Analysis of cytotoxic T cell responses to dominant and subdominant epitopes during acute and chronic lymphocytic choriomeningitis virus infection.

The cytotoxic T cell response against lymphocytic choriomeningitis virus (LCMV) in BALB/c (H-2d) mice is predominantly directed against a single immunodominant Ld-restricted epitope in the viral nucleoprotein (NP118-126). Here we report that the immunodominance of this peptide can be in part attributed to its very high affinity for Ld class I molecules. By employing motif searches and sensitive MHC class I binding assays, we also identified 5 Kd-binding peptides in the viral nucleoprotein and glycoprotein among 16 Kd motif-fitting peptides. The nucleoprotein and glycoprotein sequences also contained 18 Dd motif-fitting peptides, three of which bound Dd with weak affinity. Two of the Kd-binding peptides, residues 99-108 and residues 283-291 from the viral glycoprotein, are subdominant epitopes. Although these peptides did not sensitize target cells for direct ex vivo killing by primary antiviral CTL, secondary responses against these peptides were readily detected in BALB/c mice after acute LCMV infection. BALB/c mice that had cleared a long-term LCMV infection showed more sustained CTL responses against these subdominant epitopes, suggesting that subdominant responses might play a role in clearance of chronic infections. One of the subdominant epitopes, GP283-291, conferred partial protection against persistent viral infection after peptide vaccination.