Brian M. Sullivan

Persistent LCMV Infection Is Controlled by Blockade of Type I Interferon Signaling

INTERFER(ON)ing Persistence During persistent viral infections, a dysregulated immune response fails to control the infection. Wilson et al. (p. 202) and Teijaro et al. (p. 207; see the Perspective by Odorizzi and Wherry) show this occurs because type I interferons (IFN I), critical for early responses to viral infection, contribute to the altered immunity seen during persistent infection. Antibody blockade of IFN I signaling during chronic lymphocytic choriomeningitis virus (LCMV) in mice resulted in reduced viral titers at later stages of infection, reduced expression of inhibitory immune molecules and prevented the disruptions to secondary lymphoid organs typically observed during persistent infection with LCMV. Whether type I IFNs are also detrimental to persistent viral infection humans, such as HIV and hepatitis C virus, remains to be determined. Blockade of type I interferons leads to better control of persistent lymphocytic choriomeningitis virus infection. [Also see Perspective by Odorizzi and Wherry] During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell–dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.

Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is necessary for receptor binding, dendritic cell infection, and long-term persistence.

Arenaviruses are a major cause of hemorrhagic fevers endemic to Sub-Saharan Africa and South America, and thus a major public health and medical concern. The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is widely used as a model system for studying persistent and acute infections, as well as for gaining an understanding of mammalian immune function. When originally characterized three decades ago, the LCMV isolate, Armstrong, which causes an acute infection in adult mice, was found to differ from the LCMV Clone 13 strain that causes a persistent infection by two amino acid changes, one within the virus surface glycoprotein (GP1: F260L) and the other within the virus L polymerase (K1076Q). Mutation F260L was considered solely responsible for the exceptionally strong binding affinity of Clone 13 (L at GP1 260) to its cellular receptor, α-dystroglycan, which among cells of the immune system is preferentially expressed on dendritic cells, and consequently, alters dendritic cell function leading to viral persistence. Recently, we noted a previously overlooked nucleotide difference between these two strains that results in an additional amino acid change in GP1, N176D. To investigate the potential contribution of this newly identified mutation to the Clone 13 phenotype, we used reverse-genetics approaches to generate recombinant LCM viruses with each of these individual mutations. Phenotypic characterization of these rLCMV showed that mutation F260L, but not N176D, in the GP1 of LCMV is essential for mediating the long-term persistence of Clone 13 infections. This work emphasizes the importance of subtle differences in viral strains that determine disease outcomes.

Human Immunodeficiency Virus Type 1 Attachment, Coreceptor, and Fusion Inhibitors Are Active against both Direct and trans Infection of Primary Cells

ABSTRACT Inhibitors of human immunodeficiency virus type 1 attachment (CD4-immunoglobulin G subclass 2), CCR5 usage (PRO 140), and fusion (T-20) were tested on diverse primary cell types that represent the major targets both for infection in vivo and for the inhibition of trans infection of target cells by virus bound to dendritic cells. Although minor cell-type-dependent differences in potency were observed, each inhibitor was active on each cell type and trans infection was similarly vulnerable to inhibition at each stage of the fusion cascade.

Blockade of Interferon Beta, but Not Interferon Alpha, Signaling Controls Persistent Viral Infection

Although type I interferon (IFN-I) is thought to be beneficial against microbial infections, persistent viral infections are characterized by high interferon signatures suggesting that IFN-I signaling may promote disease pathogenesis. During persistent lymphocytic choriomeningitis virus (LCMV) infection, IFNα and IFNβ are highly induced early after infection, and blocking IFN-I receptor (IFNAR) signaling promotes virus clearance. We assessed the specific roles of IFNβ versus IFNα in controlling LCMV infection. While blockade of IFNβ alone does not alter early viral dissemination, it is important in determining lymphoid structure, lymphocyte migration, and anti-viral T cell responses that lead to accelerated virus clearance, approximating what occurs during attenuation of IFNAR signaling. Comparatively, blockade of IFNα was not associated with improved viral control, but with early dissemination of virus. Thus, despite their use of the same receptor, IFNβ and IFNα have unique and distinguishable biologic functions, with IFNβ being mainly responsible for promoting viral persistence.

Downregulation of the T-Cell Receptor Complex and Impairment of T-Cell Activation by Human Herpesvirus 6 U24 Protein

ABSTRACT We have performed a screen aimed at identifying human herpesvirus 6 (HHV-6)-encoded proteins that modulate immune recognition. Here we show that the U24 protein encoded by HHV-6 variant A downregulates cell surface expression of the T-cell receptor (TCR)/CD3 complex, a complex essential to T-cell activation and the generation of an immune adaptive response. In the presence of U24, the TCR/CD3 complex is endocytosed but is not recycled back to the plasma membrane. Instead, it accumulates in early and late endosomes. Interestingly, whereas CD3 downregulation from the cell surface is normally associated with T-cell activation, U24 downregulates CD3 independently of T-cell activation. Moreover, we found that U24-expressing T cells are resistant to activation by antigen-presenting cells. HHV-6 has evolved a unique mechanism of inhibition of T-cell activation that may impair the establishment of an adaptive immune response. Furthermore, lymphocyte activation creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. Thus, by inhibiting T-cell activation, HHV-6 might limit its reactivation and thus minimize immune recognition.

Type I interferon is a therapeutic target for virus-induced lethal vascular damage

Significance Lassa virus is, after dengue virus, the second most common cause of viral hemorrhagic fever. In susceptible individuals, Lassa virus infection is associated with vascular permeability, leading to tissue edema, organ failure, and death. Hemorrhagic fever viruses efficiently infect vascular endothelial cells, but are generally considered noncytopathic. Thus, the mechanism of virus-induced vascular injury remains unclear. Using the lymphocytic choriomeningitis virus variant clone 13, a prototype of Lassa virus, we show here that lethal vascular leakage in susceptible mice was completely prevented by type I IFN receptor blockade. Therefore, approaches that target type I IFNs or effector molecules induced by these cytokines may be considered for the treatment of Lassa fever and other severe hemorrhagic viral illnesses. The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus—a prototype of Old World arenaviruses closely related to Lassa fever virus—elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell–mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6–8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.

Slc15a4, a Gene Required for pDC Sensing of TLR Ligands, Is Required to Control Persistent Viral Infection

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.

Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV

Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein expressed on the viral surface mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1–GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated prefusion GP1–GP2 complex of the prototypic arenavirus LCMV at 3.5 Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion and illustrates the fusion regions and potential oligomeric states.

Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

The U24 Protein from Human Herpesvirus 6 and 7 Affects Endocytic Recycling

ABSTRACT Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport.