Single-cell analysis reveals divergent responses of human dendritic cells to the MVA vaccine.

Modified vaccinia Ankara (MVA) is a live, attenuated human smallpox vaccine and a vector for the development of new vaccines against infectious diseases and cancer. Efficient activation of the immune system by MVA partially relies on its encounter with dendritic cells (DCs). MVA infection of DCs leads to multiple outcomes, including cytokine production, activation of costimulatory molecules for T cell stimulation, and cell death. Here, we examined how these diverse responses are orchestrated in human DCs. Single-cell analyses revealed that the response to MVA infection in DCs was limited to early viral gene expression. In response to the early events in the viral cycle, we found that DCs grouped into three distinct clusters. A cluster of infected cells sensed the MVA genome by the intracellular innate immunity pathway mediated by cGAS, STING, TBK1, and IRF3 and subsequently produced inflammatory cytokines. In response to these cytokines, a cluster of noninfected bystander cells increased costimulatory molecule expression. A separate cluster of infected cells underwent caspase-dependent apoptosis. Induction of apoptosis persisted after inhibition of innate immunity pathway mediators independently of previously described IRF-dependent or replication-dependent pathways and was a response to early MVA gene expression. Together, our study identified multiple mechanisms that underlie the interactions of MVA with human DCs.

The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus.

Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Let me in: Control of HIV nuclear entry at the nuclear envelope.

The nuclear envelope is a physical barrier that isolates the cellular DNA from the rest of the cell, thereby limiting pathogen invasion. The Human Immunodeficiency Virus (HIV) has a remarkable ability to enter the nucleus of non-dividing target cells such as lymphocytes, macrophages and dendritic cells. While this step is critical for replication of the virus, it remains one of the less understood aspects of HIV infection. Here, we review the viral and host factors that favor or inhibit HIV entry into the nucleus, including the viral capsid, integrase, the central viral DNA flap, and the host proteins CPSF6, TNPO3, Nucleoporins, SUN1, SUN2, Cyclophilin A and MX2. We review recent perspectives on the mechanism of action of these factors, and formulate fundamental questions that remain. Overall, these findings deepen our understanding of HIV nuclear import and strengthen the favorable position of nuclear HIV entry for antiviral targeting.

Transmission of innate immune signaling by packaging of cGAMP in viral particles.

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.

Viral and cellular mechanisms of the innate immune sensing of HIV.

HIV-1 replicates in immune cells that normally respond to incoming viruses and induce antiviral immune responses. Under this constant surveillance, how HIV-1 interacts with the host to escape immune control and causes immunopathology is still being untangled. Recently, a series of HIV-1 interactions with innate sensors of viruses expressed by immune target cells have been identified. Here, we review the HIV-1 factors that escape, engage and regulate these innate immune sensors. We discuss the general principles of these interactions as well as the remarkable cell-type specificity of the regulatory mechanisms and their resulting immune responses. Innate sensors directly intersect viral replication with immunity, and understanding their triggering, or lack thereof, improves our ability to design immune interventions.

Resistance to rabies virus infection conferred by the PMLIV isoform.

Various reports implicate PML and PML nuclear bodies (NBs) in an intrinsic antiviral response targeting diverse cytoplasmic replicating RNA viruses. PML conjugation to the small ubiquitin-like modifier (SUMO) is required for its localization within NBs. PML displays antiviral effects in vivo, as PML deficiency renders mice more susceptible to infection with the rhabdovirus vesicular stomatitis virus (VSV). Cells derived from these mice are also more sensitive to infection with rabies virus, another member of the rhabdovirus family. Alternative splicing from a single gene results in the synthesis of several PML isoforms, and these are classified into seven groups, designated PMLI to -VII. We report here that expression of PMLIV or PMLIVa, which is missing exon 5, inhibited viral mRNA and protein synthesis, leading to a reduction in viral replication. However, the expression of other nuclear isoforms (PMLI to -VI) and cytoplasmic PMLVIIb failed to impair viral production. This antiviral effect required PMLIV SUMOylation, as it was not observed with PMLIV 3KR, in which the lysines involved in SUMO conjugation were mutated. Thus, PMLIV and PMLIVa may exert this isoform-specific function through interaction with specific NB protein partners via their common C-terminal region.

Dual modes of rabies P-protein association with microtubules: a novel strategy to suppress the antiviral response.

Conventional nuclear import is independent of the cytoskeleton, but recent data have shown that the import of specific proteins can be either facilitated or inhibited by microtubules (MTs). Nuclear import of the P-protein from rabies virus involves a MT-facilitated mechanism, but here, we show that P-protein is unique in that it also undergoes MT-inhibited import, with the mode of MT-interaction being regulated by the oligomeric state of the P-protein. This is the first demonstration that a protein can utilise both MT-inhibited and MT-facilitated import mechanisms, and can switch between these different modes of MT interaction to regulate its nuclear trafficking. Importantly, we show that the P-protein exploits MT-dependent mechanisms to manipulate host cell processes by switching the import of the interferon-activated transcription factor STAT1 from a conventional to a MT-inhibited mechanism. This prevents STAT1 nuclear import and signalling in response to interferon, which is vital to the host innate antiviral response. This is the first report of MT involvement in the viral subversion of interferon signalling that is central to virus pathogenicity, and identifies novel targets for the development of antiviral drugs or attenuated viruses for vaccine applications.