Andrew M. Lee

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.

IL-10 blockade facilitates DNA vaccine-induced T cell responses and enhances clearance of persistent virus infection

Therapeutic vaccination is a potentially powerful strategy to establish immune control and eradicate persistent viral infections. Large and multifunctional antiviral T cell responses are associated with control of viral persistence; however, for reasons that were mostly unclear, current therapeutic vaccination approaches to restore T cell immunity and control viral infection have been ineffective. Herein, we confirmed that neutralization of the immunosuppressive factor interleukin (IL)-10 stimulated T cell responses and improved control of established persistent lymphocytic choriomeningitis virus (LCMV) infection. Importantly, blockade of IL-10 also allowed an otherwise ineffective therapeutic DNA vaccine to further stimulate antiviral immunity, thereby increasing T cell responses and enhancing clearance of persistent LCMV replication. We therefore propose that a reason that current therapeutic vaccination strategies fail to resurrect/sustain T cell responses is because they do not alleviate the immunosuppressive environment. Consequently, blocking key suppressive factors could render ineffective vaccines more efficient at improving T cell immunity, and thereby allow immune-mediated control of persistent viral infection.

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.

Unique Small Molecule Entry Inhibitors of Hemorrhagic Fever Arenaviruses

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15–35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC50 = 500–800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC50 of 200–350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.

Site 1 Protease Is Required for Proteolytic Processing of the Glycoproteins of the South American Hemorrhagic Fever Viruses Junin, Machupo, and Guanarito

ABSTRACT The cellular proprotein convertase site 1 protease (S1P) has been implicated in the proteolytic processing of the glycoproteins (GPs) of Old World arenaviruses. Here we report that S1P is also involved in the processing of the GPs of the genetically more-distant South American hemorrhagic fever viruses Guanarito, Machupo, and Junin. Efficient cleavage of Guanarito virus GP, whose protease recognition sites deviate from the reported S1P consensus sequence, indicates a broader specificity of S1P than anticipated. Lack of GP processing of Junin virus dramatically reduced production of infectious virus and prevented cell-to-cell propagation. Infection of S1P-deficient cells resulted in viral persistence over several weeks without the emergence of escape variants able to use other cellular proteases for GP processing.

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.

Direct Evaluation of a Kinetic Model for RecA-Mediated DNA-strand exchange: The Importance of Nucleic Acid Dynamics and Entropy During Homologous Genetic Recombination

The Escherichia coli RecA protein is the prototype of a class of proteins that play central roles in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding homologous recombination. We explored the mechanism of RecA‐mediated DNA‐strand exchange using oligonucleotide substrates with the intrinsic fluorophore 6‐methylisoxanthopterin. Pre‐steady‐state spectrofluorometric analysis elucidated the earliest transient intermediates formed during recombination and delineated the mechanistic strategy by which RecA facilitates this process. The structural features of the first detectable intermediate and the energetic characteristics of its formation were consistent with interactions between a few bases of the single‐stranded DNA and the minor groove of a locally melted or stretched duplex DNA. Further analysis revealed RecA to be an unusual enzyme in that entropic rather than enthalpic contributions dominate its catalytic function, and no unambiguously active role for the protein was detected in the earliest molecular events of recombination. The data best support the conclusion that the mechanistic strategy of RecA likely relies on intrinsic DNA dynamics.

Novel approaches in anti-arenaviral drug development

Hemorrhagic fevers caused by arenaviruses are among the most devastating emerging human diseases. Considering the number of individuals affected, the current lack of a licensed vaccine, and the limited therapeutic options, arenaviruses are arguably among the most neglected tropical pathogens and the development of efficacious anti-arenaviral drugs is of high priority. Over the past years significant efforts have been undertaken to identify novel potent inhibitors of arenavirus infection. High throughput screening of small molecule libraries employing pseudotype platforms led to the discovery of several potent and broadly active inhibitors of arenavirus cell entry that are effective against the major hemorrhagic arenaviruses. Mechanistic studies revealed that these novel entry inhibitors block arenavirus membrane fusion and provided novel insights into the unusual mechanism of this process. The success of these approaches highlights the power of small molecule screens in antiviral drug discovery and establishes arenavirus membrane fusion as a robust drug target. These broad screenings have been complemented by strategies targeting cellular factors involved in productive arenavirus infection. Approaches targeting the cellular protease implicated in maturation of the fusion-active viral envelope glycoprotein identified the proteolytic processing of the arenavirus glycoprotein precursor as a novel and promising target for anti-arenaviral strategies.

Characterization of lassa virus cell entry inhibitors: Determination of the active enantiomer by asymmetric synthesis

The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the more potent (-)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer. The absolute configuration of (-)-3 was established by asymmetric synthesis of the active inhibitor (-)-(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of the prototypical inhibitors.

Pathogenesis of Lassa fever virus infection: I. Susceptibility of mice to recombinant Lassa Gp/LCMV chimeric virus

Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8(+) T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13.