Georg Kochs

The interferon-inducible myxovirus resistance proteins (Mx) belong to the dynamin-like GTPases; MxA is a restriction factor against several RNA viruses, while MxB restricts infections of lentiviruses and herpesviruses. Humans express MxA(1–662) with an N-terminal domain (NTD) of 43 residues, MxB(1–715) with an NTD of 91 residues, and a truncated MxB(26–715) with an NTD of 66 residues. Although the roles of the GTPase and stalk domains during infection are increasingly being elucidated, the functions of the different NTDs remain poorly understood. Using cell lines stably expressing Mx proteins, we show that MxB(1–715), but not MxA, inhibited infection by herpes simplex virus (HSV-1) and pseudorabies virus (PrV). Quantitative mass spectrometry and subviral fractionation experiments indicate that MxB(1–715), but not the truncated MxB(26–715) or MxA(1–662), was enriched in the tegument of HSV-1 and PrV virions. Moreover, dimeric, recombinant, chimeric proteins with the NTD peptides MxB(1–91) or MxB(1–35), and to a lesser extent MxB(26–91), bound to tegumented HSV-1 capsids, while MxA(1–43) did not. In contrast, de-tegumented HSV-1 and PrV capsids bound to proteins with MxB(1–91), but not to those with MxB(1–35) or MxA(1–43). Our findings indicate that the NTD of MxB is crucial to restrict HSV-1 and PrV infections, that newly assembled virions package MxB into the tegument around the capsid, and that both the N-terminal MxB amino acid residues 1–25 and 26–91 contribute to its binding to tegumented and de-tegumented herpesviral capsids.

Dhori virus (DHOV) ( Orthomyxoviridae, genus: Thogotovirus) is a tick-borne virus with a segmented, negative-sense RNA genome. Thogotoviruses can be divided into two clades, Thogoto-like and Dhori-like viruses, which differ in their coding strategy of the segment 6 encoded matrix protein (M): Thogoto viruses translate M from spliced transcripts and a long M (ML) from unspliced transcripts. Dhori-like viruses encode their M using unspliced transcripts, whereas no splicing or additional coding capacity has been described, yet. Here, we identified spliced transcripts of segment 6 in DHOV-infected cells by RT-PCR and sequencing. The gene product M2-248 codes for a truncated, 186 amino acids-long N-terminal moiety of M fused to 62 C-terminal amino acids from a −1 shifted reading frame. The splicing sites and the amino acid sequences of the C-terminal M2 part are conserved in Dhori-like viruses. Expression of M2-248 was confirmed in infected cells by mass spectrometry and western blot analysis and was detected in purified virions. Recombinant DHOV(ΔM2) lacking M2-248 showed reduced virulence compared with recombinant wild-type in mice but no attenuation in cell culture and no effect on virion assembly and morphology. An interferon antagonistic function of M2-248, similar to THOV ML, was excluded. Thus, our data suggest that DHOV segment 6 splicing and expression of M2-248 protein affect virulence in mammals by a mechanism distinct from THOV-ML. It remains to be analyzed how M2-248 might affect viral host switch and replication in ticks. IMPORTANCE: Dhori and Thogoto viruses are tick-transmitted orthomyxoviruses comprising two clades differing in the coding strategy of their matrix protein. The recent detection of Dhori-like Bourbon virus (BRBV) highlighted their zoonotic potential. M of Dhori-like viruses is translated from a collinear transcript of the smallest of the six genomic ssRNA segments. Thogoto-like viruses express M from spliced transcripts, and unspliced transcripts encode an extended ML protein. No analogous splicing event or additional gene product has been characterized for Dhori-like viruses, yet. Here, we describe the hitherto overlooked splicing of segment 6 transcripts of Dhori virus. The 5'-part of the processed transcript is collinear with the M-ORF, whereas splicing shifts it into a -1 frame. The predicted product M2-248 was detected in infected cells. Recombinant Dhori virus lacking M2 was attenuated in vivo , whereas replication in mammalian cells was not impaired, suggesting a modulatory function of M2 in in vivo -specific, cellular immunity-related processes.

A 48-year-old patient underwent lung transplantation because of severe COVID-19, which aggravated his underlying interstitial lung disease, despite the presence of detectable SARS-CoV-2. Subsequently, the graft is re-infected early in the post-procedural phase, leading to viral persistence for more than five months. By analyzing viral evolution and effector immune response within the transplanted organ, we observe three main findings. First, virus evolution differs in the transplanted organ compared to that in the upper respiratory tract and is affected by monoclonal SARS-CoV-2-specific antibodies and molnupiravir. Second, we show the potential clinical relevance of T cell HLA restriction that may facilitate viral clearance in the upper respiratory tract compared to the ongoing viral replication in the HLA mismatch organ. Third, close monitoring and modulation of immunosuppressive and antiviral therapy enables viral clearance in a lung transplantation setting despite incomplete SARS-CoV-2 clearance prior to transplantation.

The interferon-inducible myxovirus resistance proteins belong to the dynamin-like GTPases, with MxA being a restriction factor against several RNA viruses, while MxB restricts infections of lentiviruses and herpesviruses. Humans express MxA(1-662) with an N-terminal domain (NTD) of 43 residues, MxB(1-715) with an NTD of 91 residues, and a truncated MxB(26-715) with an NTD of 66 residues. Although the roles of the GTPase and stalk domains during infection are increasingly elucidated, the function of the different NTDs is not fully understood. Using cell lines stably expressing Mx proteins, we show that MxB(1-715) but not MxA inhibited infection with herpes simplex virus (HSV-1) and pseudorabies virus (PrV). Quantitative mass spectrometry and subviral fractionation experiments indicate that MxB(1- 715) was packaged into the tegument of HSV-1 and PrV virions, but not MxB(26-715) or MxA(1-662). Moreover, we generated recombinant proteins N-terminally fused to different NTD peptides and showed that MxB(1-91), MxB(1-35), and to a lesser extent MxB(26-91) bound to isolated HSV-1 capsids, while MxA(1-43) did not. Our findings indicate that the NTD of MxB is crucial to restrict HSV-1 and PrV infections, that newly assembled virions package MxB into the tegument around the capsid, and that the N-terminal and the C-terminal part of the MxB-NTD contribute to its binding to herpesviral capsids.

Human leukocyte antigen class I (HLA-I) molecules present intracellular peptides on the cell surface to enable CD8+ T cells to effectively control viral infections. Many viruses disrupt this antigen presentation pathway to evade immune detection. In this study, we demonstrate that SARS-CoV-2 Nsp1 impairs both the constitutive and interferon-γ (IFN-γ)-induced upregulation of HLA-I. Moreover, Nsp1 also blocks IFN-γ-induced expression of HLA-II. We found that, contrary to previously published work, the early SARS-CoV-2 B 1.1.7 Alpha variant lacking the accessory protein ORF8 retained full capacity to downregulate HLA-I, comparable to an ORF8-expressing wild-type isolate. While ectopic overexpression of ORF8 could reduce HLA-I surface levels, this effect was only observed at high expression levels. In contrast, moderate expression of the viral protein Nsp1 was sufficient to potently suppress both basal and IFN-γ-induced HLA-I, as well as HLA-II expression. To probe the underlying mechanism, we analyzed HLA-I-associated genes in previously published RNA-sequencing datasets and confirmed that Nsp1 reduces expression of components required for HLA-I biosynthesis and antigen processing. These findings identify Nsp1 as a key factor that impairs antigen presentation pathways, potentially contributing to the ability of SARS-CoV-2 to modulate immune recognition.

Antiviral restriction factors such as MxA (myxovirus resistance protein A) inhibit many viruses. Viral escape drives restriction factors to evolve rapidly at virus-binding interfaces to regain defense. Here, we explore how antiviral proteins balance restricting many viruses with evolving specificity against individual viruses. Human MxA uses its rapidly evolving loop L4 as the specificity determinant for orthomyxoviruses such as thogotovirus (THOV) and influenza (IAV). Previous combinatorial mutagenesis of rapidly evolving residues in human MxA loop L4 identified THOV “super-restrictors” and suggested an antiviral breadth-specificity trade-off. Using a modified combinatorial mutagenesis strategy, we find super-restrictor MxA variants specific to H5N1 IAV. A single L4 residue underlies the MxA breadth-specificity trade-off. However, rare “generalist” super-restrictors or a heterozygous combination of more common “specialist” super-restrictors can overcome the breadth-specificity trade-off. Our findings suggest that at least two strategies enable restriction factors such as MxA to increase their restriction of diverse viruses to overcome breadth-specificity trade-offs, which might be pervasive in host-virus conflicts.

Background: Multiple myeloma (MM) patients are at high risk of severe infections including COVID-19 due to an immune dysregulation affecting both innate and adaptive immune responses. However, our understanding of the immune responses to infection and vaccination in MM patients is limited. To gain more detailed insights into infection- and vaccine-elicited T cell immunity in MM, we studied the CD8+ T cell response on the single-epitope level in SARS-CoV-2 convalescent and mRNA-vaccinated MM patients. Methods: We compared peptide/MHC class I tetramer-enriched SARS-CoV-2-specific CD8+ T cells and antibody responses in MM patients (convalescent: n = 16, fully vaccinated: n = 5, vaccinated convalescent: n = 5) and healthy controls (HCs) (convalescent: n = 58, fully vaccinated: n = 7) either after infection with SARS-CoV-2 alone, complete mRNA vaccination or SARS-CoV-2 infection and single-shot mRNA vaccination (hybrid immunity). Results: MM patients have lower frequencies and a lower proportion of fully functional virus-specific CD8+ T cells compared to HCs, after both SARS-CoV-2 infection and vaccination. CD8+ T cell memory subset distribution in MM patients is skewed towards reduced frequencies of central memory (T_CM) T cells and higher frequencies of effector memory 1 (T_EM1) T cells. In contrast, the humoral immune response was comparable in both cohorts after viral clearance. Notably, CD8+ T cell frequencies as well as the humoral immune response were improved by a single dose of mRNA vaccine in convalescent MM patients. Conclusions: MM patients have relative immunological deficiencies in SARS-CoV-2 immunity but benefit from hybrid immunity. These findings underline the relevance of vaccinations in this vulnerable patient group.

Antiviral restriction factors such as MxA (myxovirus resistance protein A) inhibit a broad range of viruses. However, they face the challenge of maintaining this breadth as viruses evolve to escape their defense. Viral escape drives restriction factors to evolve rapidly, selecting for amino acid changes at their virus-binding interfaces to regain defense. How do restriction factors balance the breadth of antiviral functions against the need to evolve specificity against individual escaping viruses? We explored this question in human MxA, which uses its rapidly evolving loop L4 as the specificity determinant for orthomyxoviruses such as THOV and IAV. Previous combinatorial mutagenesis of rapidly evolving residues in human MxA loop L4 revealed variants with a ten-fold increase in potency against THOV. However, this strategy did not yield improved IAV restriction, suggesting a strong tradeoff between antiviral specificity and breadth. Here, using a modified combinatorial mutagenesis strategy, we find ‘super-restrictor’ MxA variants with over ten-fold enhanced restriction of the avian IAV strain H5N1 but reduced THOV restriction. Analysis of super-restrictor MxA variants reveals that the identity of residue 561 explains most of MxA’s breadth-specificity tradeoff in H5N1 versus THOV restriction. However, rare ‘generalist’ super-restrictors with enhanced restriction of both viruses allow MxA to overcome the breadth-specificity tradeoff. Finally, we show that a heterozygous combination of two ‘specialist’ super-restrictors, one against THOV and the other against IAV, enhances restriction against both viruses. Thus, two strategies enable restriction factors such as MxA to increase their restriction of diverse viruses to overcome breadth-specificity tradeoffs that may be pervasive in host-virus conflicts.

The recent discovery of Bourbon virus (BRBV) put a new focus on the genus of thogotoviruses as zoonotic, tick-transmitted pathogens within the orthomyxovirus family. Since 2014, BRBV has been linked to several human cases in the Midwest United States with severe acute febrile illness and a history of tick bites. The detection of the virus in the Lone Star tick, Amblyomma americanum, and a high sero-prevalence in wild animals suggest widespread circulation of BRBV. Phylogenetic analysis of the viral RNA genome classified BRBV into the subgroup of Dhori-like thogotoviruses. Strikingly, BRBV is apathogenic in mice, contrasting not only with the fatal disease in affected patients but also with the severe disease in mice caused by other members of the thogotovirus genus. To gain insights into this intriguing discrepancy, we will review the molecular biology and pathology of BRBV and its unique position within the thogotovirus genus. Lastly, we will discuss the zoonotic threat posed by this newly discovered pathogen.