Viroids and Viroid-like Circular RNAs: Do They Descend from Primordial Replicators?

Viroids are a unique class of plant pathogens that consist of small circular RNA molecules, between 220 and 450 nucleotides in size. Viroids encode no proteins and are the smallest known infectious agents. Viroids replicate via the rolling circle mechanism, producing multimeric intermediates which are cleaved to unit length either by ribozymes formed from both polarities of the viroid genomic RNA or by coopted host RNAses. Many viroid-like small circular RNAs are satellites of plant RNA viruses. Ribozyviruses, represented by human hepatitis delta virus, are larger viroid-like circular RNAs that additionally encode the viral nucleocapsid protein. It has been proposed that viroids are direct descendants of primordial RNA replicons that were present in the hypothetical RNA world. We argue, however, that much later origin of viroids, possibly, from recently discovered mobile genetic elements known as retrozymes, is a far more parsimonious evolutionary scenario. Nevertheless, viroids and viroid-like circular RNAs are minimal replicators that are likely to be close to the theoretical lower limit of replicator size and arguably comprise the paradigm for replicator emergence. Thus, although viroid-like replicators are unlikely to be direct descendants of primordial RNA replicators, the study of the diversity and evolution of these ultimate genetic parasites can yield insights into the earliest stages of the evolution of life.

SARS-CoV-2 Variants of Concern Infect the Respiratory Tract and Induce Inflammatory Response in Wild-Type Laboratory Mice

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern pose a major threat to public health, due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts, in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared with the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-α, IL-1β) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. In addition, robust expression of viral nucleocapsid protein and histopathological changes were detected in the lungs of B.1.351-infected mice. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.

Persistence of robust humoral immune response in COVID-19 convalescent individuals over twelve months after infection

Background SARS-CoV-2 infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). Here, we report the persistence of nAb responses over 12 months after infection despite their decreasing trend noticed from 6 months. Methods The study included sera from 497 individuals who had been infected with SARS-CoV-2 between January and August 2020. Samples were collected at 6 and 12 months after onset. The titers of IgG to the viral nucleocapsid protein (NP) and receptor-binding domain of the spike protein (RBD) were measured by CLEIA. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Results Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. While the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases (~30%) was undermined, implying the susceptibility to reinfection with the variants of concerns (VOCs). Conclusions COVID-19 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

Spatial heterogeneity of the immune compartment within the lungs of critical COVID-19 patients

Cross AR, Sansom S, Roberts I, Cerundolo L, Melero I, De Andrea C, Landecho MF, Klenerman P,Hester J, Issa F Acute respiratory distress syndrome (ARDS) is a defining feature of severe infection with theSARS-CoV-2 virus. Approaches to understand the immune response during COVID-19 are largelyconfined to characterisation of circulating leukocytes, however this approach excludes the mostrelevant cells that are active at the site of infection and injury. The aim of this study was to characterise the immune landscape across the lungs of COVID-19patients. Lung samples from three critical COVID-19 patients were assessed for histopathology,viral load, and distribution using qPCR, in situ hybridisation and immunohistochemistry.Leukocyte distribution was then assessed, and the transcript profile of selected areas examinedagainst the >1800 genes in the Cancer Transcriptome Atlas panel on the NanoString GeoMxDigital Spatial Profiling platform. Lung samples exhibited a spectrum of typical COVID-19 pathology with diffuse alveolar damageconsistent with hyaline membrane and type II pneumocyte hyperplasia, interstitialinflammation, organising pneumonia and thrombi. All tissues tested positive for SARS-CoV-2RNA using qPCR, whilst spatially resolved techniques revealed only few and sparsely distributedcells carrying the viral nucleocapsid protein. Multiplexed immunofluorescence for lymphocytes(CD3+) and macrophages (CD68+) was used to select areas of immune enrichment for spatialtranscriptomic profiling. These targeted analyses highlighted functional pathways involved inthe interferon gamma response, TCR activation and antigen presentation. Comparison acrossimmune-enriched areas identified a heterogeneity in lung infiltrates with spatial separation ofchemokine and complement production. Our data identify pathological immune pathways thatare amenable to therapeutic intervention in critical disease.

Persistence of robust humoral immune response in COVID-19 convalescent individuals over 12 months after infection

SARS-CoV-2 infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). Here we report the persistence of nAb responses over 12 months after infection despite its decreasing trend noticed from 6 months. The study included sera from 358 individuals who had been infected with SARS-CoV-2 between January and May 2020. Samples were collected at 6 and 12 months after onset. The titers of IgG to the viral nucleocapsid protein (NP) and receptor-binding domain of the spike protein (RBD) were measured by CLEIA. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. While the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases (~30%) was undermined, implying the susceptibility of reinfection to the variants of concerns (VOCs). COVID-19 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

IgG Antibodies Develop to Spike but Not to the Nucleocapsid Viral Protein in Many Asymptomatic and Light COVID-19 Cases

Since SARS-CoV-2 appeared in late 2019, many studies on the immune response to COVID-19 have been conducted, but the asymptomatic or light symptom cases were somewhat understudied as respective individuals often did not seek medical help. Here, we analyze the production of the IgG antibodies to viral nucleocapsid (N) protein and receptor-binding domain (RBD) of the spike protein and assess the serum neutralization capabilities in a cohort of patients with different levels of disease severity. In half of light or asymptomatic cases the antibodies to the nucleocapsid protein, which serve as the main target in many modern test systems, were not detected. They were detected in all cases of moderate or severe symptoms, and severe lung lesions correlated with respective higher signals. Antibodies to RBD were present in the absolute majority of samples, with levels being sometimes higher in light symptom cases. We thus suggest that the anti-RBD/anti-N antibody ratio may serve as an indicator of the disease severity. Anti-RBD IgG remained detectable after a year or more since the infection, even with a slight tendency to raise over time, and the respective signal correlated with the serum capacity to inhibit the RBD interaction with the ACE-2 receptor.

The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Antiviral RISC mainly targets viral mRNA but not genomic RNA of tospovirus

Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated, in vitro and in planta. RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic (viral and viral-complementary strands) RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group.

A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation

Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolate and profile a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who has dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the highest binding affinity mAb (nCoV396) reveals changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyperactivation analysis demonstrates that nCoV396 specifically compromises the N protein-induced complement hyperactivation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.