Attenuation of Getah virus by a single amino acid substitution at residue 253 of the E2 protein that might be part of a new heparan sulfate binding site on alphaviruses

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo . Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the 3D structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks also progress less consistently in cells depleted for SAF-A, contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed two distinct effects of SAF-A depletion: first, the boundaries between early- and late-replicating domains become more blurred; and second, SAF-A depletion causes replication timing changes that tend to bring regions of discordant domain compartmentalisation and replication timing into concordance. Associated with these defects, SAF-A-depleted cells show elevated -H2AX formation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

SAFA initiates innate immunity against cytoplasmic RNA virus SFTSV infection

Nuclear scaffold attachment factor A (SAFA) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS with a high fatality rate of up to 30%. It remains elusive whether and how cytoplasmic SFTSV can be sensed by the RNA sensor SAFA. Here, we demonstrated that SAFA was able to detect SFTSV infection and mediate antiviral interferon and inflammatory responses. Transcription and expression levels of SAFA were strikingly upregulated under SFTSV infection. SAFA was retained in the cytoplasm by interaction with SFTSV nucleocapsid protein (NP). Importantly, SFTSV genomic RNA was recognized by cytoplasmic SAFA, which recruited and promoted activation of the STING-TBK1 signaling axis against SFTSV infection. Of note, the nuclear localization signal (NLS) domain of SAFA was important for interaction with SFTSV NP and recognition of SFTSV RNA in the cytoplasm. In conclusion, our study reveals a novel antiviral mechanism in which SAFA functions as a novel cytoplasmic RNA sensor that directly recognizes RNA virus SFTSV and mediates an antiviral response.

Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA

During brain homeostasis, stem cell fate determination is crucial to guarantee function, adaptation and regeneration while preventing neurodegeneration and cognitive impairment. How neural stem cells (NSCs) are instructed to generate neurons or glia is not well understood. Here we addressed how fate is resolved in multipotent adult hippocampal NSCs, and identify Scaffold Attachment Factor B1 (Safb1) as a determinant of neuron production by blocking glial commitment. Safb1 is sufficient to block oligodendrocytic differentiation of NSCs by preventing expression of the transcription factor NFIB at the post-transcriptional level. Detailed interrogation of the Drosha interactome and functional validation revealed that Safb1 enhances NFIB mRNA cleavage in a Drosha-dependent fashion. Thus, our study provides a cellular mechanism for selective NSC fate regulation by post-transcriptional destabilization of mRNAs. Given the importance of NSC maintenance and fate determination in the adult brain, our findings have major implications for cell-specific gene expression, brain disease and aging.

Evolution of the GII.3[P12] Norovirus from 2010 to 2019 in Jiangsu, China

Objectives Norovirus genotype GII.3[P12] strains have been an important pathogen for sporadic gastroenteritis infection. In previous studies of GII.3[P12], the number of specimens and time span are relatively small, which is difficult to truly reflect the infection and evolution of this type of norovirus. Here we report a molecular epidemiological study of the NoVs prevalent in Jiangsu between 2010 and 2019 to investigate the evolution of the GII.3[P12] strains in China. Methods In this study 60 GII.3[P12] norovirus strains were sequenced and analyzed for evolution, recombination, and selection pressure using bioanalysis software. Results The GII.3[P12] strains were continuously detected during the study period, which showed a high constituent ratio in males, in winter and among children aged 0–11 months, respectively. A time-scaled evolutionary tree showed that both GII.P12 RdRp and GII.3 VP1 sequences were grouped into three major clusters (Cluster I–III). Most GII.3[P12] strains were mainly located in sub-cluster (SC) II of Cluster III. A SimPlot analysis identified GII.3[P12] strain to be as an ORF1-intragenic recombinant of GII.4[P12] and GII.3[P21]. The RdRp genes of the GII.3[P12] showed a higher mean substitution rate than those of all GII.P12, while the VP1 genes of the GII.3[P12] showed a lower mean substitution rate than those of all GII.3. Alignment of the GII.3 capsid sequences revealed that three HBGA binding sites of all known GII.3 strains remained conserved, while several amino acid mutations in the predicted antibody binding sites were detected. The mutation at 385 was within predicted antibody binding regions, close to host attachment factor binding sites. Positive and negative selection sites were estimated. Two common positively selected sites (sites 385 and 406) were located on the surface of the protruding domain. Moreover, an amino acid substitution (aa204) was estimated to be near the active site of the RdRp protein. Conclusions We conducted a comprehensive analysis on the epidemic and evolution of GII.3[P12] noroviruses and the results suggested that evolution was possibly driven by intergenic recombination and mutations in some key amino acid sites.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

Novel Cell-Penetrating Peptides Derived From Scaffold-Attachment- Factor A Inhibits Cancer Cell Proliferation and Survival

Scaffold-attachment-factor A (SAFA) has important roles in many normal and pathologic cellular processes but the scope of its function in cancer cells is unknown. Here, we report dominant-negative activity of novel peptides derived from the SAP and RGG-domains of SAFA and their effects on proliferation, survival and the epigenetic landscape in a range of cancer cell types. The RGG-derived peptide dysregulates SAFA binding and regulation of alternatively spliced targets and decreases levels of key spliceosome proteins in a cell-type specific manner. In contrast, the SAP-derived peptide reduces active histone marks, promotes chromatin compaction, and activates the DNA damage response and cell death in a subset of cancer cell types. Our findings reveal an unprecedented function of SAFA-derived peptides in regulating diverse SAFA molecular functions as a tumor suppressive mechanism and demonstrate the potential therapeutic utility of SAFA-peptides in a wide range of cancer cells.

Evolution of the GII.3[P12] Norovirus from 2010 to 2019 in Jiangsu, China

Objectives: Norovirus genotype GII.3[P12] strains have been an important pathogen for sporadic gastroenteritis infection. In previous studies of GII.3[P12], the number of specimens and time span are relatively small, which is difficult to truly reflect the infection and evolution of this type of norovirus. Here we report a molecular epidemiological study of the NoVs prevalent in Jiangsu between 2010 and 2019 to investigate the evolution of the GII.3[P12] strains in China. Methods: In this study 60 GII.3[P12] norovirus strains were sequenced and analyzed for evolution, recombination, and selection pressure using bioanalysis software. Results: The GII.3[P12] strains were continuously detected during the study period, which showed a high constituent ratio in males, among children aged 0–11 months and winter. A time-scaled evolutionary tree showed that both GII.P12 RdRp and GII.3 VP1 sequences were grouped into three major clusters (Cluster I-III). Most GII.3[P12] strains were mainly located in sub-cluster (SC) II of Cluster III. A SimPlot analysis identified GII.3[P12] strain to be as an ORF1-intragenic recombinant of GII.4[P12] and GII.3[P21]. The RdRp genes of the GII.3[P12] showed a higher mean substitution rate than those of all GII.P12, while the VP1 genes of the GII.3[P12] showed a lower mean substitution rate than those of all GII.3. Alignment of the GII.3 capsid sequences revealed that three HBGA binding sites of all known GII.3 strains remained conserved, while several amino acid mutations in the predicted antibody binding sites were detected. The mutation at 385 was within predicted antibody binding regions, close to host attachment factor binding sites. Positive and negative selection sites were estimated. Two common positively selected sites (sites 385 and 406) were located on the surface of the protruding domain. Moreover, an amino acid substitution (aa204) was estimated to be near the active site of the RdRp protein. These results suggested that evolution of the GII.3[P12] noroviruses was possibly driven by intergenic recombination and mutations in some key amino acid sites. Conclusions: The GII.3[P12] recombinant strain was dominant for the GII.P12 and GII.3 noroviruses in China, which is therefore a major concern for causing gastrointestinal infection.