Persistent paramyxovirus infections: in co-infections the parainfluenza virus type 5 persistent phenotype is dominant over the lytic phenotype.

Parainfluenza virus type 5 (PIV5) can either have a persistent or a lytic phenotype in cultured cells. We have previously shown that the phenotype is determined by the phosphorylation status of the phosphoprotein (P). Single amino acid substitutions at critical residues, including a serine-to-phenylalanine substitution at position 157 on P, result in a switch between persistent and lytic phenotypes. Here, using PIV5 vectors expressing either mCherry or GFP with persistent or lytic phenotypes, we show that in co-infections the persistent phenotype is dominant. Thus, in contrast to the cell death observed with cells infected solely with the lytic variant, in co-infected cells persistence is immediately established and both lytic and persistent genotypes persist. Furthermore, 10-20 % of virus released from dually infected cells contains both genotypes, indicating that PIV5 particles can package more than one genome. Co-infected cells continue to maintain both genotypes/phenotypes during cell passage, as do individual colonies of cells derived from a culture of persistently infected cells. A refinement of our model on how the dynamics of virus selection may occur in vivo is presented.

Partial human Janus kinase 1 deficiency predominantly impairs responses to interferon gamma and intracellular control of mycobacteria.

Purpose: Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/ß and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control. Methods: We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA. Results: Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts. Conclusion: Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors.

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2.

Excessive type I interferon (IFNα/ß) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/ß, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/ß signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/ß signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/ß activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit.

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.

Genome Sequence of the Parainfluenza Virus 5 Strain That Persistently Infects AGS Cells.

We have sequenced the parainfluenza virus 5 strain that persistently infects the commonly used AGS human cell line without causing cytopathology. This virus is most closely related to human strains, indicating that it may have originated from biopsy material or from laboratory contamination during generation of the cell line.

Human IFNAR2 deficiency: Lessons for antiviral immunity.

Type I interferon (IFN-α/ß) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/ß in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis after inoculation of the live attenuated measles, mumps, and rubella (MMR) vaccine. By targeted resequencing, we identified a homozygous mutation in the high-affinity IFN-α/ß receptor (IFNAR2) in the proband, as well as a newborn sibling, that rendered cells unresponsive to IFN-α/ß. Reconstitution of the proband's cells with wild-type IFNAR2 restored IFN-α/ß responsiveness and control of IFN-attenuated viruses. Despite the severe outcome of systemic live vaccine challenge, the proband had previously shown no evidence of heightened susceptibility to respiratory viral pathogens. The phenotype of IFNAR2 deficiency, together with similar findings in STAT2-deficient patients, supports an essential but narrow role for IFN-α/ß in human antiviral immunity.

STAT2 deficiency and susceptibility to viral illness in humans.

Severe infectious disease in children may be a manifestation of primary immunodeficiency. These genetic disorders represent important experiments of nature with the capacity to elucidate nonredundant mechanisms of human immunity. We hypothesized that a primary defect of innate antiviral immunity was responsible for unusually severe viral illness in two siblings; the proband developed disseminated vaccine strain measles following routine immunization, whereas an infant brother died after a 2-d febrile illness from an unknown viral infection. Patient fibroblasts were indeed abnormally permissive for viral replication in vitro, associated with profound failure of type I IFN signaling and absence of STAT2 protein. Sequencing of genomic DNA and RNA revealed a homozygous mutation in intron 4 of STAT2 that prevented correct splicing in patient cells. Subsequently, other family members were identified with the same genetic lesion. Despite documented infection by known viral pathogens, some of which have been more severe than normal, surviving STAT2-deficient individuals have remained generally healthy, with no obvious defects in their adaptive immunity or developmental abnormalities. These findings imply that type I IFN signaling [through interferon-stimulated gene factor 3 (ISGF3)] is surprisingly not essential for host defense against the majority of common childhood viral infections.

STAT-1- and IRF-3-dependent pathways are not essential for repression of ICP0-null mutant herpes simplex virus type 1 in human fibroblasts.

Efficient herpes simplex virus type 1 (HSV-1) infection of human fibroblasts (HFs) is highly dependent on the viral immediate-early regulatory protein ICP0 unless the infection is conducted at a high multiplicity. ICP0-null mutant HSV-1 exhibits a plaque-forming defect of up to 3 orders of magnitude in HFs, whereas in many other cell types, this defect varies between 10- and 30-fold. The reasons for the high ICP0 requirement for HSV-1 infection in HFs have not been established definitively. Previous studies using other cell types suggested that ICP0-null mutant HSV-1 is hypersensitive to interferon and that this sensitivity is dependent on the cellular promyelocytic leukemia (PML) protein. To investigate the roles of two important aspects of interferon signaling in the phenotype of ICP0-null mutant HSV-1, we isolated HFs depleted of STAT-1 or interferon regulatory factor 3 (IRF-3). Surprisingly, plaque formation by the mutant virus was not improved in either cell type. We found that the sensitivity to interferon pretreatment of both ICP0-null mutant and wild-type (wt) HSV-1 was highly dependent on the multiplicity of infection. At a low multiplicity in virus yield experiments, both viruses were extremely susceptible to interferon pretreatment of HFs, but the sensitivity of the wild type but not the mutant could be overcome at higher multiplicities. We found that both wt and ICP0-null mutant HSV-1 remained sensitive to interferon in PML-depleted HFs albeit to an apparently lesser extent than in control cells. The data imply that the substantial reduction in ICP0-null HSV-1 infectivity at a low multiplicity in HFs does not occur through the activities of STAT-1- and IRF-3-dependent pathways and cannot be explained solely by enhanced sensitivity to interferon. We suggest that antiviral activities induced by interferon may be separable from and additive to those resulting from PML-related intrinsic resistance mechanisms.