γ-herpesvirus latency attenuates Mycobacterium tuberculosis infection in mice.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a bacterial pathogen which is transmitted via aerosol and establishes a chronic lung infection. In naïve hosts, Mtb grows for several weeks without being restricted by IFNγ-producing T cells, which eventually accumulate and limit Mtb dissemination. In this study, we used a mouse model of Mtb/γ-herpesvirus (γHV) coinfection to test the hypothesis that latent γHV infection alters host resistance to Mtb. γHVs are DNA viruses which elicit a polyclonal T cell response and attenuate some acute bacterial pathogens in mice; whether γHVs modulate infection with Mtb is unknown. Here, mice harboring latent mouse gammaherpesvirus 68 (MHV68)-a γHV genetically and biologically related to human Epstein Barr virus (EBV)-were infected via aerosol with a low dose of virulent Mtb. Mtb burdens and IFNγ+ T cell frequencies in mice with latent MHV68 (MHV68POS mice) were subsequently measured and compared to control mice that did not harbor latent MHV68 (MHV68NEG mice). Relative to MHV68NEG controls, MHV68POS mice more effectively limited Mtb growth and dissemination, and had higher frequencies of CD4+IFNγ+ cells in lung-draining lymph nodes. Collectively, our results support a model wherein latent γHV confers moderate protection against subsequent Mtb infection.

A DNA-sensing-independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB-mediated innate immunity against DNA virus infection.

DExD/H-box helicase 9 (DHX9), or RNA helicase A (RHA), is an abundant multifunctional nuclear protein. Although it was previously reported to act as a cytosolic DNA sensor in plasmacytoid dendritic cells (pDCs), the role and molecular mechanisms of action of DHX9 in cells that are not pDCs during DNA virus infection are not clear. Here, a macrophage-specific knockout and a fibroblast-specific knockdown of DHX9 impaired antiviral innate immunity against DNA viruses, leading to increased virus replication. DHX9 enhanced NF-κB-mediated transactivation in the nucleus, which required its ATPase-dependent helicase (ATPase/helicase) domain, but not the cytosolic DNA-sensing domain. In addition, DNA virus infection did not induce cytoplasmic translocation of nuclear DHX9 in macrophages and fibroblasts. Nuclear DHX9 was associated with a multiprotein complex including both NF-κB p65 and RNA polymerase II (RNAPII) in chromatin containing NF-κB-binding sites. DHX9 was essential for the recruitment of RNAPII rather than NF-κB p65, to the corresponding promoters; this function also required its ATPase/helicase activity. Taken together, our results show a critical role of nuclear DHX9 (as a transcription coactivator) in the stimulation of NF-κB-mediated innate immunity against DNA virus infection, independently of DHX9's DNA-sensing function.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Regulation of gammaherpesvirus lytic replication by endoplasmic reticulum stress-induced transcription factors ATF4 and CHOP.

The stress-induced unfolded protein response (UPR) in the endoplasmic reticulum (ER) involves various signaling cross-talks and controls cell fate. B-cell receptor (BCR) signaling, which can trigger UPR, induces gammaherpesvirus lytic replication and serves as a physiological mechanism for gammaherpesvirus reactivation in vivo However, how the UPR regulates BCR-mediated gammaherpesvirus infection is unknown. Here, we demonstrate that the ER stressors tunicamycin and thapsigargin inhibit BCR-mediated murine gammaherpesvirus 68 (MHV68) lytic replication by inducing expression of the UPR mediator Bip and blocking activation of Akt, ERK, and JNK. Both Bip and the downstream transcription factor ATF4 inhibited BCR-mediated MHV68 lytic gene expression, whereas UPR-induced C/EBP homologous protein (CHOP) was required for and promoted BCR-mediated MHV68 lytic replication by suppressing upstream Bip and ATF4 expression. Bip knockout was sufficient to rescue BCR-mediated MHV68 lytic gene expression in CHOP knockout cells, and this rescue was blocked by ectopic ATF4 expression. Furthermore, ATF4 directly inhibited promoter activity of the MHV68 lytic switch transactivator RTA. Altogether, we show that ER stress-induced CHOP inhibits Bip and ATF4 expression and that ATF4, in turn, plays a critical role in CHOP-mediated regulation of BCR-controlled MHV68 lytic replication. We conclude that ER stress-mediated UPR and BCR signaling pathways are interconnected and form a complex network to regulate the gammaherpesvirus infection cycle.

Interferon Regulatory Factor 1 and Type I Interferon Cooperate To Control Acute Gammaherpesvirus Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection in >95% of adults worldwide and are associated with a variety of malignancies. Coevolution of gammaherpesviruses with their hosts has resulted in an intricate relationship between the virus and the host immune system, and perturbation of the virus-host balance results in pathology. Interferon regulatory factor 1 (IRF-1) is a tumor suppressor that is also involved in the regulation of innate and adaptive immune responses. Here, we show that type I interferon (IFN) and IRF-1 cooperate to control acute gammaherpesvirus infection. Specifically, we demonstrate that a combination of IRF-1 and type I IFN signaling ensures host survival during acute gammaherpesvirus infection and supports IFN gamma-mediated suppression of viral replication. Thus, our studies reveal an intriguing cross talk between IRF-1 and type I and II IFNs in the induction of the antiviral state during acute gammaherpesvirus infection. IMPORTANCE: Gammaherpesviruses establish chronic infection in a majority of adults, and this long-term infection is associated with virus-driven development of a range of malignancies. In contrast, a brief period of active gammaherpesvirus replication during acute infection of a naive host is subclinical in most individuals. Here, we discovered that a combination of type I interferon (IFN) signaling and interferon regulatory factor 1 (IRF-1) expression is required to ensure survival of a gammaherpesvirus-infected host past the first 8 days of infection. Specifically, both type I IFN receptor and IRF-1 expression potentiated antiviral effects of type II IFN to restrict gammaherpesvirus replication in vivo, in the lungs, and in vitro, in primary macrophage cultures.

In vivo activation of toll-like receptor-9 induces an age-dependent abortive lytic cycle reactivation of murine gammaherpesvirus-68.

Infection of mice with murine gammaherpesvirus-68 (γHV-68) serves as a model to understand the pathogenesis of persistent viral infections, including the potential for co-infections to modulate viral latency. We have previously found that infection of neonates (8-day-old mice) with γHV-68 resulted in a high level of persistence of the virus in the lungs as well as the spleen, in contrast to infection of adult mice, for which long-term latency was only readily detected in the spleen. In this study we investigated whether stimulation of toll-like receptor (TLR)9 would modulate viral latency in mice infected with γHV-68 in an age-dependent manner. Pups and adult mice were injected with the synthetic TLR9 ligand CpG ODN at 30 dpi, at which time long-term latency has been established. Three days after CpG injection, the lungs and spleens were removed, and a limiting dilution assay was done to determine the frequency of latently infected cells. RNA was extracted to measure viral transcripts using a ribonuclease protection assay. We observed that CpG injection resulted in an increase in the frequency of latently-infected cells in both the lungs and spleens of infected pups, but only in the spleens of infected adult mice. No preformed virus was detected, suggesting that TLR9 stimulation did not trigger complete viral reactivation. When we examined viral gene expression in these same tissues, we observed expression only of the immediate early lytic genes, rta and K3, but not the early DNA polymerase gene or late gB transcript indicative of an abortive reactivation in the spleen. Additionally, mice infected as pups had greater numbers of germinal center B cells in the spleen following CpG injection, whereas CpG stimulated the expansion of follicular zone B cells in adult mice. These data suggest that stimulation of TLR9 differentially modulates gammaherpesvirus latency via an age-dependent mechanism.

A gammaherpesvirus complement regulatory protein promotes initiation of infection by activation of protein kinase Akt/PKB.

BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein.

Characterization of a novel wood mouse virus related to murid herpesvirus 4.

Two novel gammaherpesviruses were isolated, one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter, designated wood mouse herpesvirus (WMHV), was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved, especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus, Brest herpesvirus (BRHV), which was isolated ostensibly from a white-toothed shrew (Crocidura russula), was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus, WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4, except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.

Open reading frame 33 of a gammaherpesvirus encodes a tegument protein essential for virion morphogenesis and egress.

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.

Induction of tachykinin production in airway epithelia in response to viral infection.

BACKGROUND: The tachykinins are implicated in neurogenic inflammation and the neuropeptide substance P in particular has been shown to be a proinflammatory mediator. A role for the tachykinins in host response to lung challenge has been previously demonstrated but has been focused predominantly on the release of the tachykinins from nerves innervating the lung. We have previously demonstrated the most dramatic phenotype described for the substance P encoding gene preprotachykinin-A (PPT-A) to date in controlling the host immune response to the murine gammaherpesvirus 68, in the lung. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have utilised transgenic mice engineered to co-ordinately express the beta-galactosidase marker gene along with PPT-A to facilitate the tracking of PPT-A expression. Using a combination of these mice and conventional immunohistology we now demonstrate that PPT-A gene expression and substance P peptide are induced in cells of the respiratory tract including tracheal, bronchiolar and alveolar epithelial cells and macrophages after viral infection. This induction was observed 24h post infection, prior to observable inflammation and the expression of pro-inflammatory chemokines in this model. Induced expression of the PPT-A gene and peptide persisted in the lower respiratory tract through day 7 post infection. CONCLUSIONS/SIGNIFICANCE: Non-neuronal PPT-A expression early after infection may have important clinical implications for the progression or management of lung disease or infection aside from the well characterised later involvement of the tachykinins during the inflammatory response.

Ecstasy (3,4-methylenedioxymethamphetamine) limits murine gammaherpesvirus-68 induced monokine expression.

While Ecstasy (3,4-methylenedioxymethamphetamine, MDMA) has been shown to modulate immune responses, no studies have addressed drug-induced alterations to viral infection. In this study, bone marrow-derived macrophages were exposed to MDMA, then infected with murine gammaherpesvirus-68, and the expression of monokines assessed. MDMA-induced reductions in virus-stimulated monokine mRNA expression were observed in a dose-dependent manner. In particular, IL-6 mRNA expression and secretion was significantly decreased in gammaherpesvirus-infected macrophages exposed to MDMA. Concentrations of MDMA capable of reducing monokine production did not induce significant cell death and allowed normal viral gene expression. These studies represent the first to demonstrate the ability of this drug of abuse to alter a viral-induced macrophage response.

Recombinant murine gammaherpesvirus 68 (MHV-68) as challenge virus to test efficacy of vaccination against chronic virus infections in the mouse model.

Efficient vaccines against AIDS, Hepatitis C and other persistent virus infections are urgently needed. Vaccine development has been especially hampered by the lack of suitable small animal models to reliably test the protective capacity of candidate vaccines against such chronic viral infections. A natural mouse pathogen such as MHV-68 that persists lifelong after infection, appears to be a particularly promising candidate for a more relevant model system. Here, we investigated infections with recombinant MHV-68 as novel mouse challenge model to test the efficacy of heterologous vaccines based on recombinant modified vaccinia virus Ankara (MVA). To apply ovalbumin (OVA) as a model antigen, we constructed the recombinant virus MHV-68-OVA by BAC technology and characterized genetic stability and replicative capacity of the virus in vitro and in vivo. We demonstrated the ability of MHV-68-OVA to produce ovalbumin upon tissue culture infection. Moreover, the use of MHV-68-OVA-infected target cells allowed for efficient ex vivo amplification of OVA-specific, MHC class I-restricted CD8 T cells derived from MVA-OVA-vaccinated C57BL/6 mice. Finally, we immunized C57BL/6 mice with MVA-OVA and challenged the animals with MHV-68-OVA testing different time points and routes of infection. Vaccinated mice were infected with MHV-68-OVA but showed reduced viral loads in the acute and latent phase of challenge infection. These data strongly suggest the usefulness of the MHV-68 challenge model for further evaluation of recombinant vaccines against persisting virus infections.

Differential transcription of ovine herpesvirus 2 genes in lymphocytes from reservoir and susceptible species.

Ovine herpesvirus 2 (OvHV-2) is a lymphotropic gammaherpesvirus that asymptomatically infects most sheep, but causes malignant catarrhal fever in cattle, bison, pigs and deer. There is no permissive cell culture system but OvHV-2-infected T lymphocytes can be cultured from diseased animals. We showed that the OvHV-2 genome was in a circular conformation in sheep peripheral blood mononuclear cells and that the latency-associated ORF73 was transcribed, while expression of the productive cycle genes ORF9 (DNA polymerase) and ORF50 (R-transactivator) was barely detectable, suggestive of latency. Doxorubicin treatment of these cells induced the appearance of linear viral DNA and transcription of productive cycle genes along with several viral unique genes. In contrast, cultured T cells from diseased cattle and rabbits contained a mixture of circular and linear genome configurations indicative of a mixture of latently- and productively-infected cells. Most of the OvHV-2 unique genes were transcribed in these cells but ORF50 expression was only seen after doxorubicin treatment indicating a 'leaky' latent pattern of gene expression. 5-azacytidine treatment increased the proportion of circular DNA and inhibited the expression of most of the OvHV-2 unique genes except Ov2.5 (vIL-10) and Ov4.5 (Bcl-2 homologue) in the cattle cell line. These studies provide key insights into the differences in OvHV-2 gene expression in cells from reservoir and susceptible species and, for the first time, an in vitro system for studying the latent and productive phases of the OvHV-2 virus life cycle.

Forced lytic replication impairs host colonization by a latency-deficient mutant of murine gammaherpesvirus-68.

A regulated switch between latent and lytic gene expression is common to all known herpesviruses. However, the effects on host colonization of altering this switch are largely unknown. We deregulated the transcription of the gene encoding the major lytic transactivator of murine gammaherpesvirus-68, ORF50, by inserting a new and powerful promoter element in its 5' untranslated region. In vitro, the mutant virus (M50) transcribed ORF50 at a high level and showed more rapid lytic spread in permissive fibroblast cultures, but in vivo, the M50 virus showed a severe deficit in latency establishment, with no sign of the infectious mononucleosis-like illness normally associated with wild-type infection. Although a low level of M50 viral DNA was detectable by PCR in spleens, replication-competent virus could not be recovered beyond 10 days post-infection. The M50 virus was also attenuated in immunocompromised mice. Thus a gammaherpesvirus unable to shut off lytic cycle gene expression showed severely restricted host colonization.

Differential gamma-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice.

Murine gamma-herpesvirus 68 (MHV-68) provides an important experimental model for analyzing gamma-herpesvirus latent infection. After intranasal infection with MHV-68, we analyzed the distribution of the virus in different anatomical locations and purified populations of cells. Our data show that long-term latency is maintained in a variety of anatomical locations and cell populations with different frequencies. Importantly, we demonstrate that although latency in the lung is established in a variety of cell subsets, long-term latency in the lung is only maintained in B cells. In contrast, splenic latency is maintained in macrophages and dendritic cells, as well as in B cells. In blood, isotype-switched B cells constitute the major viral reservoir. These results show that the cell subsets in which latency is established vary within different anatomical sites. Finally, we demonstrate that long-term latency is accompanied by a low level of infectious virus in lung and spleen. These data have important implications for understanding the establishment and maintenance of latency by gamma(2)-herpesviruses.

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus.

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.

Antibody-mediated control of persistent gamma-herpesvirus infection.

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.

Identification of the in vivo role of a viral bcl-2.

Many gamma-herpesviruses encode candidate oncogenes including homologues of host bcl-2 and cyclin proteins (v-bcl-2, v-cyclin), but the physiologic roles of these genes during infection are not known. We show for the first time in any virus system the physiologic role of v-bcl-2. A gamma-herpesvirus v-bcl-2 was essential for efficient ex vivo reactivation from latent infection, and for both persistent replication and virulence during chronic infection of immunocompromised (interferon [IFN]-gamma(-/-)) mice. The v-cyclin was also critical for the same stages in pathogenesis. Strikingly, while the v-bcl-2 and v-cyclin were important for chronic infection, these genes were not essential for viral replication in cell culture, viral replication during acute infection in vivo, establishment of latent infection, or virulence during acute infection. We conclude that v-bcl-2 and v-cyclin have important roles during latent and persistent gamma-herpesvirus infection and that herpesviruses encode genes with specific roles during chronic infection and disease, but not acute infection and disease. As gamma-herpesviruses primarily cause human disease during chronic infection, these chronic disease genes may be important targets for therapeutic intervention.

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus.

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8(+) T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-gamma production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Natural history of murine gamma-herpesvirus infection.

Murine gamma-herpesvirus 68 (MHV-68) is a natural pathogen of small rodents and insectivores (mice, voles and shrews). The primary infection is characterized by virus replication in lung epithelial cells and the establishment of a latent infection in B lymphocytes. The virus is also observed to persist in lung epithelial cells, dendritic cells and macrophages. Splenomegaly is observed two weeks after infection, in which there is a CD4+ T-cell-mediated expansion of B and T cells in the spleen. At three weeks post-infection an infectious mononucleosis-like syndrome is observed involving a major expansion of Vbeta4+CD8+ T cells. Later in the course of persistent infection, ca. 10% of mice develop lymphoproliferative disease characterized as lymphomas of B-cell origin. The genome from MHV-68 strain g2.4 has been sequenced and contains ca. 73 genes, the majority of which are collinear and homologous to other gamma-herpesviruses. The genome includes cellular homologues for a complement-regulatory protein, Bcl-2, cyclin D and interleukin-8 receptor and a set of novel genes M1 to M4. The function of these genes in the context of latent infections, evasion of immune responses and virus-mediated pathologies is discussed. Both innate and adaptive immune responses play an active role in limiting virus infection. The absence of type I interferon (IFN) results in a lethal MHV-68 infection, emphasizing the central role of these cytokines at the initial stages of infection. In contrast, type II IFN is not essential for the recovery from infection in the lung, but a failure of type II IFN receptor signalling results in the atrophy of lymphoid tissue associated with virus persistence. Splenic atrophy appears to be the result of immunopathology, since in the absence of CD8+ T cells no pathology occurs. CD8+ T cells play a major role in recovery from the primary infection, and also in regulating latently infected cells expressing the M2 gene product. CD4+ T cells have a key role in surveillance against virus recurrences in the lung, in part mediated through 'help' in the genesis of neutralizing antibodies. In the absence of CD4+ T cells, virus-specific CD8+ T cells are able to control the primary infection in the respiratory tract, yet surprisingly the memory CD8+ T cells generated are unable to inhibit virus recurrences in the lung. This could be explained in part by the observations that this virus can downregulate major histocompatibility complex class I expression and also restrict inflammatory cell responses by producing a chemokine-binding protein (M3 gene product). MHV-68 provides an excellent model to explore methods for controlling gamma-herpesvirus infection through vaccination and chemotherapy. Vaccination with gp150 (a homologue of gp350 of Epstein-Barr virus) results in a reduction in splenomegaly and virus latency but does not block replication in the lung, nor the establishment of a latent infection. Even when lung virus infection is greatly reduced following the action of CD8+ T cells, induced via a prime-boost vaccination strategy, a latent infection is established. Potent antiviral compounds such as the nucleoside analogue 2'deoxy-5-ethyl-beta-4'-thiouridine, which disrupts virus replication in vivo, cannot inhibit the establishment of a latent infection. Clearly, devising strategies to interrupt the establishment of latent virus infections may well prove impossible with existing methods.