Biology subjects

Forrest, J. C.

Publications and source records attributed to Forrest, J. C..

10 recordsLinked to original sources

bioRxiv2026

Single cell in vivo analysis of type I IFN and NK cell-mediated control of B cell infection densities during acute gammaherpesvirus infection

Immunodeficient patients are at risk of severe complications following infection with the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposis sarcoma-associated herpesvirus (KSHV). Due to the strict host specificity of human gammaherpesviruses, murine gammaherpesvirus 68 (MHV68) is widely used as an in vivo model to study gammaherpesvirus pathogenesis. While type I interferons (IFNs) and natural killer (NK) cells are known to contribute to antiviral defense during MHV68 infection, how these innate immune mechanisms control infection within lymphoid tissues at the level of individual infected cells remains incompletely understood. Here, we used an MHV68-DsRed reporter virus to visualize infected cells in the lymph nodes of wildtype and type I IFN receptor-deficient (Ifnar1-/-) mice by flow cytometry, immunohistochemistry, and two-photon microscopy. We found that type I IFN signaling plays a major role in limiting both local viral infection in draining lymph nodes and systemic dissemination to the spleen during acute infection. In the absence of IFNAR signaling, infected B cells accumulated to higher numbers, including an increased proportion of infected germinal center phenotype B cells, and this was accompanied by enhanced activation of T and B lymphocytes. Using two-photon microscopy, we further examined NK cell behavior within infected lymph nodes. NK cells were rapidly recruited to sites of infection but did not form stable clusters or prolonged contacts with infected cells. Nevertheless, NK cell depletion resulted in increased numbers of infected cells, indicating that NK cells contribute to the control of acute MHV68 infection despite the absence of detectable long-lasting interactions with infected cells. Together, our findings provide a single-cell view of acute gammaherpesvirus infection and innate immune control within lymph nodes in vivo. These data refine our understanding of how type I IFN responses and NK cells restrict early gammaherpesvirus spread and shape infection dynamics within lymphoid tissues. Author SummaryCertain viruses can remain in the body for life and cause little harm in healthy individuals. However, when the immune system is weakened, these infections can lead to cancer or other life-threatening diseases. To study human gammaherpesviruses within the living body, scientists often use a closely related mouse virus to understand how the immune system controls these infections. In this study, we tracked virus-infected cells within lymph nodes, important sites of the immune responses. Using a fluorescent virus together with advanced imaging techniques, we visualized infected cells directly in intact tissues. We found that type I interferons, a key component of the early antiviral defense, strongly limited the number of infected cells. Mice that could not respond to type I interferons developed substantially larger infections. We also investigated the role of natural killer cells, immune cells that provide rapid protection against viral infections. Although these cells quickly accumulated at sites of infection, they rarely formed prolonged contacts with infected cells. Together, our findings provide a detailed view of how innate immune defenses restrict gammaherpesvirus infection within lymph nodes. By revealing how early immune responses limit viral spread, this work improves our understanding of mechanisms that protect against severe herpesvirus-associated disease.

immunology↗

bioRxiv2026

The amplitude of gammaherpesvirus lytic replication dictates adaptive immune activation: Potential implications for KSHV LANA in immune evasion

Adaptive immune responses to primary Kaposi sarcoma-associated herpesvirus (KSHV) infection are poorly defined. To develop better small-animal models for understanding KSHV pathogenesis and immunity, we previously generated a chimeric virus in which the KSHV latency-associated nuclear antigen (kLANA), a conserved multifunctional protein critical for viral latency, was exchanged for the LANA homolog in murine gammaherpesvirus 68 (MHV68). Despite comparable levels of latent infection between WT and KLKI MHV68, kLANA directly repressed MHV68 lytic replication and reactivation. We therefore hypothesized that suppression of lytic replication by kLANA dampens adaptive immune responses. To test this, mice were infected with equivalent doses of either WT or KLKI MHV68 and adaptive immune responses were evaluated over time. B and T cell activation was starkly reduced following KLKI MHV68 infection, despite a potent virus-specific effector CD8+ T cell response against both viruses. These phenotypes were independent of inoculating dose, as high dose infection with KLKI MHV68 still showed reduced adaptive immune cell activation. In contrast, infection of Ifnar1-/-mice, which support enhanced KLKI MHV68 lytic replication, led to potent adaptive cellular and humoral immune activation by both WT and KLKI viruses, suggesting that the level of viral replication, and not simply amount of virus present, is a major driver of adaptive immunity during GHV infection. Collectively, these data support the hypothesis that kLANA-mediated suppression of lytic replication facilitates immune evasion by holding viral replication below a threshold for potent induction of adaptive immunity. IMPORTANCEKSHV is a gammaherpesvirus that establishes lifelong, chronic infections in humans and increases the risk of virus-associated cancers. Currently, there is little information on how primary KSHV infection influences adaptive immune development in healthy individuals. Rodent models, such as murine gammaherpesvirus 68 (MHV68), provide a valuable laboratory system for studying gammaherpesvirus pathogenesis in vivo. In this study, we report that infection with a previously characterized chimeric KSHV-MHV68 virus expressing KSHV LANA represses lytic viral replication and elicits weak antiviral adaptive immune responses following primary infection, despite efficient latency establishment. Using this chimeric MHV68 virus, we demonstrate that lytic viral amplification must breach a threshold to trigger a potent virus-specific adaptive immune response. We propose that KSHV, through LANA, evades detection by repressing lytic viral replication to remain "below the radar" of adaptive immune defenses during host colonization.

microbiology↗

bioRxiv2024

Vaccination with a Replication-Dead Murine Gammaherpesvirus Lacking Viral Pathogenesis Genes Inhibits WT Virus Infection

Gammaherpesviruses are oncogenic pathogens that establish lifelong infections. There are no FDA-approved vaccines against Epstein-Barr virus or Kaposi sarcoma herpesvirus. Murine gammaherpesvirus-68 (MHV68) infection of mice provides a system for investigating of gammaherpesvirus pathogenesis and testing vaccine strategies. Prime-boost vaccination with a replication-dead virus (RDV) that does not express the essential replication and transactivator protein (RTA) encoded by ORF50 (RDV-50.stop) protected against WT virus replication and reduce latency in C57BL/6 mice and prevented lethal disease in Ifnar1-/- mice. To further improve the RDV vaccine and more closely model KSHV vaccine design, we generated an RDV lacking the unique M1-M4 genes and the non-coding tRNA-miRNA-encoded RNAs (TMERs) 6, 7, and 8 that collectively promote latency of MHV68 in vivo. Prime-boost vaccination of mice with RDV-50.stop{Delta}M1-M4 elicited neutralizing antibodies and virus-specific CD8 T-cell responses in lungs and spleens, the respective sites of acute replication and latency, that were comparable to RDV-50.stop vaccination. When challenged with WT MHV68, vaccinated mice exhibited a near-complete block of lytic replication and a reduction in latency and reactivation. We conclude that major determinants of MHV68 pathogenesis are not required components for eliciting a protective immune response.

microbiology↗

bioRxiv2023

Intrinsic p53 Activation Restricts Gammaherpesvirus-Driven Germinal Center B Cell Expansion during Latency Establishment

Gammaherpesviruses (GHV) are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center (GC) B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of GHV pathogenesis, we demonstrate in vivo that tumor suppressor p53 is activated specifically in B cells that are latently infected by MHV68. In the absence of p53, the early expansion of MHV68 latency was greatly increased, especially in GC B cells, a cell-type whose proliferation was conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of GC B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that EBV-encoded latent membrane protein 1 (LMP1) similarly triggers a p53 response in primary B cells. Our data highlight a model in which GHV latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53. IMPORTANCEGammaherpesviruses cause lifelong infections of their hosts, commonly referred to as latency, that can lead to cancer. Latency establishment benefits from the functions of viral proteins that augment and amplify B cell activation, proliferation, and differentiation signals. In uninfected cells, off-schedule cellular differentiation would typically trigger anti-proliferative responses by effector proteins known as tumor suppressors. However, tumor suppressor responses to gammaherpesvirus manipulation of cellular processes remain understudied, especially those that occur during latency establishment in a living organism. Here we identify p53, a tumor suppressor commonly mutated in cancer, as a host factor that limits virus-driven B cell proliferation and differentiation, and thus, viral colonization of a host. We demonstrate that p53 activation occurs in response to viral latency proteins that induce B cell activation. This work informs a gap in our understanding of intrinsic cellular defense mechanisms that restrict lifelong GHV infection.

microbiology↗

bioRxiv2023

Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice

Gammaherpesviruses (GHVs) are oncogenic viruses that establish lifelong infections and are significant causes of human morbidity and mortality. While several vaccine strategies to limit GHV infection and disease are in development, there are no FDA-approved vaccines for human GHVs. As a new approach to gammaherpesvirus vaccination, we developed and tested a replication-dead virus (RDV) platform, using murine gammaherpesvirus 68 (MHV68), a well-established mouse model for gammaherpesvirus pathogenesis studies and preclinical therapeutic evaluations. We employed codon-shuffling-based complementation to generate revertant-free RDV lacking expression of the essential replication and transactivator protein (RTA) encoded by ORF50 to arrest viral gene expression early after de novo infection. Inoculation with RDV-50.stop exposes the host to intact virion particles and leads to limited lytic gene expression in infected cells. Prime-boost vaccination of mice with RDV-50.stop elicited virus-specific neutralizing antibody and effector T cell responses in the lung and spleen. Vaccination with RDV-50.stop resulted in a near complete abolishment of virus replication in the lung 7 days post-challenge and virus reactivation from spleen 16 days post-challenge with WT MHV68. Ifnar1-/- mice, which lack the type I interferon receptor, exhibit severe disease upon infection with WT MHV68. RDV-50.stop vaccination of Ifnar1-/- mice prevented wasting and mortality upon challenge with WT MHV68. These results demonstrate that prime-boost vaccination with a GHV that is unable to undergo lytic replication offers protection against acute replication, reactivation, and severe disease upon WT virus challenge. ImportanceGammaherpesviruses establish chronic infections that place a host at life-long risk for the development of lymphoproliferative disorders. While these viruses are endemic within the adult human population, there are currently no FDA-approved vaccines available to prevent acute infection or the later onset of lymphoproliferative disorders. We report here the use of a prime-boost vaccination strategy utilizing replication-dead virus strains to protect mice from acute disease following challenge with WT virus. We demonstrate that vaccination with replication-dead viruses is both safe and effective within immunocompetent and immunodeficient mice, and protects from viral replication, reactivation, and onset of severe disease.

microbiology↗

bioRxiv2023

Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian Uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect, unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF encoded by ORF59. MHV68 vUNG colocalized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone, or in combination. Last, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCEGammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus associated cancers.

microbiology↗

bioRxiv2023

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/fmice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses. IMPORTANCEThere are no directed therapies to the latency program of the gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor STAT3 is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

microbiology↗

bioRxiv2021

Lytic Replication and Reactivation from B cells Is Not Required for Maintaining Gammaherpesvirus Latency in vivo

Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced, however the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Stimulation of polyclonal B cell activation and proliferation by treating mice with lipopolysaccharide (LPS), which promotes MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir. Together, these data demonstrate that lytic replication in B cells is not required for MHV68 latency establishment and maintenance and further indicate that B cell proliferation, and not reactivation per se, is a major mechanism for maintaining latent viral genomes in the host. IMPORTANCEGammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within immune cells.

microbiology↗

bioRxiv2020

p53 Controls Murine Gammaherpesvirus Latency and Prevents Infection-Associated IgH/c- Myc Translocations

Gammaherpesviruses (GHVs) establish life-long infections and cause cancer in humans and other animals. To facilitate chronic infection, GHV oncoproteins promote cellular proliferation and differentiation. Aberrant cell-cycle progression driven by viral oncogenes should trigger activation of tumor suppressor p53, unless p53 is functionally deactivated during GHV latency establishment. However, interactions of GHVs with the p53 pathway during the establishment and maintenance of latent infection are poorly defined. Here we demonstrate in vivo that p53 is induced specifically in infected cells during latency establishment by murine gammaherpesvirus 68 (MHV68). In the absence of p53, MHV68 latency establishment was significantly increased, especially in germinal center B cells, and correlated with enhanced cellular proliferation. However, enhanced latency was not sustainable, and MHV68 exhibited a defect in long-term latency maintenance in p53-deficient mice. Moreover, IgH/c-Myc translocations were readily detected in B cells from infected p53-null mice indicating virus-driven genomic instability. These data demonstrate that p53 intrinsically restricts MHV68 latency establishment and reveal a paradigm in which a host restriction factor provides a long-term benefit to a chronic pathogen by limiting infection-associated damage.

microbiology↗

bioRxiv2020

Deletion of Murine Gammaherpesvirus Gene M2 in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish life-long, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immune compromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, neither colonization of draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization. IMPORTANCEGammaherpesviruses establish life-long chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

microbiology↗