Activation and Evasion of Innate Immunity by Gammaherpesviruses.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the vast majority of adults worldwide. Importantly, these viruses are associated with numerous malignancies and are responsible for significant human cancer burden. These virus-associated cancers are due, in part, to the ability of gammaherpesviruses to successfully evade the innate immune response throughout the course of infection. In this review, we will summarize the current understanding of how gammaherpesviruses are detected by innate immune sensors, how these viruses evade recognition by host cells, and how this knowledge can inform novel therapeutic approaches for these viruses and their associated diseases.

Gammaherpesvirus Usurps Host IL-17 Signaling To Support the Establishment of Chronic Infection.

Gammaherpesviruses establish lifelong infection and are associated with a variety of cancers, including B cell lymphomas. These viruses manipulate the B cell differentiation process to establish lifelong infection in memory B cells. Specifically, gammaherpesviruses infect naive B cells and promote entry of both infected and uninfected naive B cells into germinal centers, where the virus usurps rapid proliferation of germinal center B cells to exponentially increase its cellular latent reservoir. In addition to facilitating the establishment of latent infection, germinal center B cells are thought to be the target of viral transformation. In this study, we have uncovered a novel proviral role of host interleukin 17A (IL-17A), a well-established antibacterial and antifungal factor. Loss of IL-17A signaling attenuated the establishment of chronic gammaherpesvirus infection and gammaherpesvirus-driven germinal center response in a route of inoculation-dependent manner. Further, IL-17A treatment directly supported gammaherpesvirus reactivation and de novo lytic infection. This study is the first demonstration of a multifaceted proviral role of IL-17 signaling.IMPORTANCE Gammaherpesviruses establish lifelong infections in a majority of humans and are associated with B cell lymphomas. IL-17A is a host cytokine that plays a well-established role in the clearance of bacterial and fungal infections; however, the role of IL-17A in viral infections is poorly understood. In this study, we show that IL-17A signaling promoted the establishment of chronic gammaherpesvirus infection following the mucosal route of infection, viral lytic replication, and reactivation from latency. Thus, our study unveils a novel proviral role of IL-17A signaling in gammaherpesvirus infection.

Interferon Regulatory Factor 3 Supports the Establishment of Chronic Gammaherpesvirus Infection in a Route- and Dose-Dependent Manner.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with several malignancies, including B cell lymphomas. Uniquely, these viruses manipulate B cell differentiation to establish long-term latency in memory B cells. This study focuses on the interaction between gammaherpesviruses and interferon regulatory factor 3 (IRF-3), a ubiquitously expressed transcription factor with multiple direct target genes, including beta interferon (IFN-ß), a type I IFN. IRF-3 attenuates acute replication of a plethora of viruses, including gammaherpesvirus. Furthermore, IRF-3-driven IFN-ß expression is antagonized by the conserved gammaherpesvirus protein kinase during lytic virus replication in vitro In this study, we have uncovered an unexpected proviral role of IRF-3 during chronic gammaherpesvirus infection. In contrast to the antiviral activity of IRF-3 during acute infection, IRF-3 facilitated establishment of latent gammaherpesvirus infection in B cells, particularly, germinal center and activated B cells, the cell types critical for both natural infection and viral lymphomagenesis. This proviral role of IRF-3 was further modified by the route of infection and viral dose. Furthermore, using a combination of viral and host genetics, we show that IRF-3 deficiency does not rescue attenuated chronic infection of a protein kinase null gammaherpesvirus mutant, highlighting the multifunctional nature of the conserved gammaherpesvirus protein kinases in vivo In summary, this study unveils an unexpected proviral nature of the classical innate immune factor, IRF-3, during chronic virus infection.IMPORTANCE Interferon regulatory factor 3 (IRF-3) is a critical component of the innate immune response, in part due to its transactivation of beta interferon (IFN-ß) expression. Similar to that observed in all acute virus infections examined to date, IRF-3 suppresses lytic viral replication during acute gammaherpesvirus infection. Because gammaherpesviruses establish lifelong infection, this study aimed to define the antiviral activity of IRF-3 during chronic infection. Surprisingly, we found that, in contrast to acute infection, IRF-3 supported the establishment of gammaherpesvirus latency in splenic B cells, revealing an unexpected proviral nature of this classical innate immune host factor.

Interferon Regulatory Factor 7 Attenuates Chronic Gammaherpesvirus Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with a variety of malignancies, including lymphomas. Interferon regulatory factor 7 (IRF-7) is an innate immune transcription factor that restricts acute replication of diverse viruses, including murine gammaherpesvirus 68 (MHV68). Importantly, very little is known about the role of IRF-7 during chronic virus infections. In this study, we demonstrate that IRF-7 attenuates chronic infection by restricting establishment of gammaherpesvirus latency in the peritoneal cavity and, to a lesser extent, viral reactivation in the spleen. Despite the classical role of IRF-7 as a stimulator of type I interferon (IFN) transcription, there were no global effects on the expression of IFN-induced genes (ISGs) in the absence of IRF-7, with only a few ISGs showing attenuated expression in IRF-7-deficient peritoneal cells. Further, IRF-7 expression was dispensable for the induction of a virus-specific CD8 T cell response. In contrast, IRF-7 expression restricted latent gammaherpesvirus infection in the peritoneal cavity under conditions where the viral latent reservoir is predominantly hosted by peritoneal B cells. This report is the first demonstration of the antiviral role of IRF-7 during the chronic stage of gammaherpesvirus infection.IMPORTANCE The innate immune system of the host is critical for the restriction of acute viral infections. In contrast, the role of the innate immune network during chronic herpesvirus infection remains poorly defined. Interferon regulatory factor 7 (IRF-7) is a transcription factor with many target genes, including type I interferons (IFNs). In this study, we show that the antiviral role of IRF-7 continues into the chronic phase of gammaherpesvirus infection, wherein IRF-7 restricts the establishment of viral latency and viral reactivation. This study is, to our knowledge, the first to define the role of IRF-7 in chronic virus infection.

Innate immunity and alpha/gammaherpesviruses: first impressions last a lifetime.

Innate immune system is considered the first line of defense during viral invasion, with the wealth of the literature demonstrating innate immune control of diverse viruses during acute infection. What is far less clear is the role of innate immune system during chronic virus infections. This short review focuses on alphaherpesviruses and gammaherpesviruses, two highly prevalent herpesvirus subfamilies that, following a brief, once in a lifetime period of acute lytic infection, establish life-long latent infection that is characterized by sporadic reactivation in an immunocompetent host. In spite of many similarities, these two viral families are characterized by distinct cellular tropism and pathogenesis. Here we focus on the published in vivo studies to review known interactions of these two viral subfamilies with the innate immunity of the intact host, both during acute and, particularly, chronic virus infection.

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire.

A common biologic property of the gammaherpesviruses Epstein-Barr Virus and Kaposi sarcoma herpesvirus is their use of B lymphocytes as a reservoir of latency in healthy individuals that can undergo oncogenic transformation later in life. Gammaherpesviruses (GHVs) employ an impressive arsenal of proteins and non-coding RNAs to reprogram lymphocytes for proliferative expansion. Within lymphoid tissues, the germinal center (GC) reaction is a hub of B cell proliferation and death. The goal of a GC is to generate and then select for a pool of immunoglobulin (Ig) genes that will provide a protective humoral adaptive immune response. B cells infected with GHVs are detected in GCs and bear the hallmark signatures of the mutagenic processes of somatic hypermutation and isotype class switching of the Ig genes. However, data also supports extrafollicular B cells as a reservoir engaged by GHVs. Next-generation sequencing technologies provide unprecedented detail of the Ig sequence that informs the natural history of infection at the single cell level. Here, we review recent reports from human and murine GHV systems that identify striking differences in the immunoglobulin repertoire of infected B cells compared to their uninfected counterparts. Implications for virus biology, GHV-associated cancers, and host immune dysfunction will be discussed.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

Insights into CD8 T Cell Activation and Exhaustion from a Mouse Gammaherpesvirus Model.

(S.R.S.) I was introduced to viral immunology while working in Peter Doherty's laboratory in the early stages of my research career, inspiring a lifelong interest in this area. During those early years under Peter's mentorship, we studied a mouse gammaherpesvirus model (murine gammaherpesvirus-68 [MHV-68]) that provided a useful small animal model for investigating the immunological control of gammaherpesvirus infection. Interestingly, while CD4 T cells were not required for acute control of MHV-68 in the lung, CD8 T cell-mediated control was progressively lost in the absence of CD4 T cell help, leading to viral recrudescence. This was one of several early studies showing that CD8 T cell control of persistent viral infections was lost in the absence of CD4 T cell help, preceding the concept of CD8 T cell exhaustion. Further studies showed that MHV-68 infection of mice offered a unique model for comparing the mechanisms of acute and long-term control of a persistent viral infection and developing strategies for reversing T cell exhaustion. Here, we provide a brief review of the literature on CD8 T cell activation and exhaustion in this model, focusing on the role of CD40 and B7 family members and including some previously unpublished data.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Limited protection against γ-herpesvirus infection by replication-deficient virus particles.

The γ-herpesviruses have proved hard to vaccination against, with no convincing protection against long-term latent infection by recombinant viral subunits. In experimental settings, whole-virus vaccines have proved more effective, even when the vaccine virus itself establishes latent infection poorly. The main alternative is replication-deficient virus particles. Here high-dose, replication-deficient murid herpesvirus-4 only protected mice partially against wild-type infection. By contrast, latency-deficient but replication-competent vaccine protected mice strongly, even when delivered non-invasively to the olfactory epithelium. Thus, this approach seems to provide the best chance of a safe and effective γ-herpesvirus vaccine.

Gammaherpesviruses and B Cells: A Relationship That Lasts a Lifetime.

Gammaherpesviruses are highly prevalent pathogens that establish life-long infection and are associated with diverse malignancies, including lymphoproliferative diseases and B cell lymphomas. Unlike other viruses that either do not infect B cells or infect B cells transiently, gammaherpesviruses manipulate physiological B cell differentiation to establish life-long infection in memory B cells. Disruption of such viral manipulation by genetic or environmental causes is likely to seed viral lymphomagenesis. In this review, we discuss physiological and unique host and viral mechanisms usurped by gammaherpesviruses to fine tune host B cell biology for optimal infection establishment and maintenance.

A CD4+ T Cell-NK Cell Axis of Gammaherpesvirus Control.

CD4+ T cells are essential to control herpesviruses. Murid herpesvirus 4 (MuHV-4)-driven lung disease in CD4+ T-cell-deficient mice provides a well-studied example. Protective CD4+ T cells have been hypothesized to kill infected cells directly. However, removing major histocompatibility complex class II (MHCII) from LysM+ or CD11c+ cells increased MuHV-4 replication not in those cells but in type 1 alveolar epithelial cells, which lack MHCII, LysM, or CD11c. Disruption of MHCII in infected cells had no effect. Therefore, CD4+ T cells engaged uninfected presenting cells and protected indirectly. Mice lacking MHCII in LysM+ or CD11c+ cells maintained systemic antiviral CD4+ T cell responses, but recruited fewer CD4+ T cells into infected lungs. NK cell infiltration was also reduced, and NK cell depletion normalized infection between MHCII-deficient and control mice. Therefore, NK cell recruitment seemed to be an important component of CD4+ T-cell-dependent protection. Disruption of viral CD8+ T cell evasion made this defense redundant, suggesting that it is important mainly to control CD8-evasive pathogens.IMPORTANCE Gammaherpesviruses are widespread and cause cancers. CD4+ T cells are a key defense. We found that they defend indirectly, engaging uninfected presenting cells and recruiting innate immune cells to attack infected targets. This segregation of CD4+ T cells from immediate contact with infection helps the immune system to cope with viral evasion. Priming this defense by vaccination offers a way to protect against gammaherpesvirus-induced cancers.

The Role of Dendritic Cells in Immune Control and Vaccination against -Herpesviruses.

The two human oncogenic -herpesviruses, Epstein Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), are prototypic pathogens that are controlled by T cell responses. Despite their ubiquitous distribution, persistent infections and transforming potential, most carriers' immune systems control them for life. Therefore, they serve as paradigms of how near-perfect cell-mediated immune control can be initiated and maintained for decades. Interestingly, EBV especially quite efficiently avoids dendritic cell (DC) activation, and little evidence exists that these most potent antigen-presenting cells of the human body are involved in the priming of immune control against this tumor virus. However, DCs can be harnessed therapeutically to expand virus-specific T cells for adoptive transfer therapy of patients with virus-associated malignancies and are also currently explored for vaccinations. Unfortunately, despite 55 and 25 years of research on EBV and KSHV, respectively, the priming of their immune control that belongs to the most robust and durable immune responses in humans still remains unclear.

Stem cell transplantation impairs dendritic cell trafficking and herpesvirus immunity.

Long-term survivors after hematopoietic stem cell transplantation are at high risk of infection, which accounts for one-third of all deaths related to stem cell transplantation. Little is known about the cause of inferior host defense after immune cell reconstitution. Here, we exploited a murine syngeneic BM transplantation (BMT) model of late infection with murine gammaherpesvirus 68 (MHV-68) to determine the role of conventional DC (cDC) trafficking in adaptive immunity in BMT mice. After infection, the expression of chemokine Ccl21 in the lung is reduced and the migration of cDCs into lung draining lymph nodes (dLNs) is impaired in BMT mice, limiting the opportunity for cDCs to prime Th cells in the dLNs. While cDC subsets are redundant in priming Th1 cells, Notch2 functions in cDC2s are required for priming increased Th17 responses in BMT mice, and cDC1s can lessen this activity. Importantly, Th17 cells can be primed both in the lungs and dLNs, allowing for increased Th17 responses without optimum cDC trafficking in BMT mice. Taken together, impaired cDC trafficking in BMT mice reduces protective Th1 responses and allows increased pathogenic Th17 responses. Thus, we have revealed a previously unknown mechanism for BMT procedures to cause long-term inferior immune responses to herpes viral infection.

A randomised vaccine field trial in Kenya demonstrates protection against wildebeest-associated malignant catarrhal fever in cattle.

Wildebeest-associated malignant catarrhal fever (WA-MCF), a fatal disease of cattle caused by alcelaphine herpesvirus 1 (AlHV-1), is one of the most important seasonal diseases of cattle in wildebeest endemic areas, with annual incidence reaching 10%. Here we report efficacy of over 80% for a vaccine based on the attenuated AlHV-1 C500 strain, in preventing fatal WA-MCF in cattle exposed to natural wildebeest challenge. The study was conducted at Kapiti Plains Ranch Ltd, south-east of Nairobi, Kenya. In 2016, 146 cattle were selected for a randomised placebo-controlled trial. Cattle were stratified according to breed and age and randomly assigned to groups given vaccine or culture medium mixed with Emulsigen®. Cattle received prime and boost inoculations one month apart and few adverse reactions (n = 4) were observed. Indirect ELISA demonstrated that all cattle in the vaccine group developed a serological response to AlHV-1. The study herd was grazed with wildebeest from one month after booster vaccination. Three cattle, two that received vaccine and one control, succumbed to conditions unrelated to WA-MCF before the study ended. Twenty-five cattle succumbed to WA-MCF; four of the remaining 71 cattle in the vaccine group (5.6%) and 21 of the remaining 72 control cattle (29.2%; χ2 = 13.6, df = 1, p < 0.001). All of the WA-MCF affected cattle were confirmed by PCR to be infected with AlHV-1 and in 23 cases exhibited histopathology typical of WA-MCF. Vaccine efficacy was determined to be 80.6% (95% CI 46.5-93.0%). Hence, the AlHV-1 C500 vaccine is a safe and potentially effective novel method for controlling WA-MCF in cattle. The implementation of this vaccine may have significant impacts on marginalised cattle keeping communities.

High-Dimensional Characterization of IL-10 Production and IL-10-Dependent Regulation during Primary Gammaherpesvirus Infection.

IL-10 is a potent immunomodulatory cytokine produced by multiple cell types to restrain immune activation. Many herpesviruses use the IL-10 pathway to facilitate infection, but how endogenous IL-10 is regulated during primary infection in vivo remains poorly characterized. In this study, we infected mice with murine gammaherpesvirus 68 (γHV68) and analyzed the production and genetic contribution of IL-10 by mass cytometry (cytometry by time-of-flight) analysis. γHV68 infection elicited a breadth of effector CD4 T cells in the lungs of acutely infected mice, including a highly activated effector subset that coexpressed IFN-γ, TNF-α, and IL-10. By using IL-10 GFP transcriptional reporter mice, we identified that IL-10 was primarily expressed within CD4 T cells during acute infection in the lungs. IL10gfp-expressing CD4 T cells were highly proliferative and characterized by the expression of multiple coinhibitory receptors, including PD-1 and LAG-3. When we analyzed acute γHV68 infection of IL-10-deficient mice, we found that IL-10 limits the frequency of both myeloid and effector CD4 T cell subsets in the infected lung, with minimal changes at a distant mucosal site. These data emphasize the unique insights that high-dimensional analysis can afford in investigating antiviral immunity and provide new insights into the breadth, phenotype, and function of IL-10-expressing effector CD4 T cells during acute virus infection.

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.

The effect of maternal immunity on the equine gammaherpesvirus type 2 and 5 viral load and antibody response.

Two types of gammaherpesviruses (γEHV) are known to infect horses, EHV-2 and EHV-5. Foals become infected early in life, probably via the upper respiratory tract, despite maternal antibodies. In this study, we analyzed samples from a herd of mares and their foals. The foals were followed from birth to 22 months of age and the dams during the first 6 months postpartum. Blood and nasal swab samples were taken regularly for evaluation of antibody responses, virus isolation and viral load by qPCR. EHV-2 was isolated on day 5, and EHV-5 on day 12, earlier than previously reported. γEHV specific antibodies were not detectable in serum of foals before colostrum intake but peaked a few days after colostrum. Overall, EHV-2 viral load peaked in nasal swab at three to four months of age, paralleled with decline in maternal antibodies, but EHV-5 viral load did not peak until month 12. Maternal antibodies had a notable effect on the viral load and induction of endogenous antibody production. Foals were grouped in two groups depending on the mare's γEHV specific total IgG levels in serum at birth, group-high and group-low. Group-high had higher levels of maternal γEHV specific total IgG and IgG4/7 for the first 3 months, but when the endogenous production had superseded maternal antibodies, group-low was higher. The maternal antibodies had an effect on the γEHV viral load. Group-low peaked in EHV-2 viral load one month earlier than group-high. These effects were more evident for EHV-5, as there were seven months between the viral load peaks for the groups. The study provides information on how maternal antibody transfer affects γEHV shedding and antibody production in offspring. It also extends our knowledge on the occurrence of EHV-2 and EHV-5 infection in foals during the first two years of life.

Neuropilin-1 Regulates the Secondary CD8 T Cell Response to Virus Infection.

Neuropilin-1 (Nrp1) plays important roles in axonal guidance in neurons and in the growth of new blood vessels. There is also a growing appreciation for roles played by neuropilin-1 in the immune response. This molecule is important for the function of regulatory T cells; however, roles in other T cell populations have not been identified. Here, we show that neuropilin-1 is expressed during the peak of the antiviral CD8 T cell response during murine gammaherpesvirus infection. Using a conditional knockout model, we deleted Nrp1 either before infection or after CD8 T cell memory had been established. We found that deletion of Nrp1 skewed the acute CD8 T cell response toward a memory precursor-like phenotype; however, the ensuing resting memory response was similar regardless of Nrp1 expression. Interestingly, Nrp1 deletion had differing effects on the recall response depending on the timing of deletion. When deleted before infection, Nrp1 deficiency inhibited the secondary response. Deletion just prior to reexposure to virus led to an enhanced secondary response. Interestingly, these effects were observed only in mice infected with a persistent strain of murine gammaherpesvirus and not with a nonpersistent mutant strain. These data highlight a multifaceted role for neuropilin-1 in memory CD8 T cell differentiation, dependent upon the stage of the T cell response and characteristics of the infectious agent. Several therapeutic anticancer therapies focus on inhibition of Nrp1 to restrict tumor growth, and so knowledge of how Nrp1 blockade may affect the CD8 T cell response will provide a better understanding of treatment consequences.IMPORTANCE CD8 T cell responses are critical to control both virus infections and tumors. The ability of these cells to persist for long periods of time can result in lifelong immunity, as relatively small populations of cells can expand rapidly to counter reexposure to the same insult. Understanding the molecules necessary for this rapid secondary expansion is critical if we are to develop therapies that can provide lifelong protection. This report shows an important and complex role for the molecule neuropilin-1 in the secondary response. Several cancer therapies targeting neuropilin-1 are in development, and this work will lead to better understanding of the effect these therapies could have upon the protective CD8 T cell response.