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Journal of Virology Jul 2024Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to...
UNLABELLED
Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase . Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3C-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection.
IMPORTANCE
RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.
PubMed: 38953667
DOI: 10.1128/jvi.00498-24 -
Applied and Environmental Microbiology Jul 2024To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm...
UNLABELLED
To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The Δ mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal -regulatory element embedded in the neighboring upstream gene negatively regulates expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA.
IMPORTANCE
Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.
PubMed: 38953660
DOI: 10.1128/aem.00741-24 -
Journal of Virology Jul 2024
Correction for Huang et al., "p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories".
PubMed: 38953642
DOI: 10.1128/jvi.00920-24 -
MBio Jul 2024Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their...
Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system . To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.
PubMed: 38953638
DOI: 10.1128/mbio.01445-24 -
MBio Jul 2024Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in...
UNLABELLED
Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an model to advance studies of infant-specific diseases and drug discovery for this population.
IMPORTANCE
Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.
PubMed: 38953637
DOI: 10.1128/mbio.01316-24 -
MBio Jul 2024The continued evolution of severe acute respiratory syndrome 2 (SARS-CoV-2) requires persistent monitoring of its subvariants. Omicron subvariants are responsible for...
The continued evolution of severe acute respiratory syndrome 2 (SARS-CoV-2) requires persistent monitoring of its subvariants. Omicron subvariants are responsible for the vast majority of SARS-CoV-2 infections worldwide, with XBB and BA.2.86 sublineages representing more than 90% of circulating strains as of January 2024. To better understand parameters involved in viral transmission, we characterized the functional properties of Spike glycoproteins from BA.2.75, CH.1.1, DV.7.1, BA.4/5, BQ.1.1, XBB, XBB.1, XBB.1.16, XBB.1.5, FD.1.1, EG.5.1, HK.3, BA.2.86 and JN.1. We tested their capacity to evade plasma-mediated recognition and neutralization, binding to angiotensin-converting enzyme 2 (ACE2), their susceptibility to cold inactivation, Spike processing, as well as the impact of temperature on Spike-ACE2 interaction. We found that compared to the early wild-type (D614G) strain, most Omicron subvariants' Spike glycoproteins evolved to escape recognition and neutralization by plasma from individuals who received a fifth dose of bivalent (BA.1 or BA.4/5) mRNA vaccine and improve ACE2 binding, particularly at low temperatures. Moreover, BA.2.86 had the best affinity for ACE2 at all temperatures tested. We found that Omicron subvariants' Spike processing is associated with their susceptibility to cold inactivation. Intriguingly, we found that Spike-ACE2 binding at low temperature was significantly associated with growth rates of Omicron subvariants in humans. Overall, we report that Spikes from newly emerged Omicron subvariants are relatively more stable and resistant to plasma-mediated neutralization, present improved affinity for ACE2 which is associated, particularly at low temperatures, with their growth rates.IMPORTANCEThe persistent evolution of SARS-CoV-2 gave rise to a wide range of variants harboring new mutations in their Spike glycoproteins. Several factors have been associated with viral transmission and fitness such as plasma-neutralization escape and ACE2 interaction. To better understand whether additional factors could be of importance in SARS-CoV-2 variants' transmission, we characterize the functional properties of Spike glycoproteins from several Omicron subvariants. We found that the Spike glycoprotein of Omicron subvariants presents an improved escape from plasma-mediated recognition and neutralization, Spike processing, and ACE2 binding which was further improved at low temperature. Intriguingly, Spike-ACE2 interaction at low temperature is strongly associated with viral growth rate, as such, low temperatures could represent another parameter affecting viral transmission.
PubMed: 38953636
DOI: 10.1128/mbio.00907-24 -
MBio Jul 2024Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus...
Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating broad viral tropism and diversified druggable potential. We and others have found that antihistamine drugs, particularly histamine receptor H1 (HRH1) antagonists, potently inhibit SARS-CoV-2 infection. In this study, we provided compelling evidence that HRH1 acts as an alternative receptor for SARS-CoV-2 by directly binding to the viral spike protein. HRH1 also synergistically enhanced hACE2-dependent viral entry by interacting with hACE2. Antihistamine drugs effectively prevent viral infection by competitively binding to HRH1, thereby disrupting the interaction between the spike protein and its receptor. Multiple inhibition assays revealed that antihistamine drugs broadly inhibited the infection of various SARS-CoV-2 mutants with an average IC50 of 2.4 µM. The prophylactic function of these drugs was further confirmed by authentic SARS-CoV-2 infection assays and humanized mouse challenge experiments, demonstrating the therapeutic potential of antihistamine drugs for combating coronavirus disease 19.IMPORTANCEIn addition to human angiotensin-converting enzyme 2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can utilize alternative cofactors to facilitate viral entry. In this study, we discovered that histamine receptor H1 (HRH1) not only functions as an independent receptor for SARS-CoV-2 but also synergistically enhances ACE2-dependent viral entry by directly interacting with ACE2. Further studies have demonstrated that HRH1 facilitates the entry of SARS-CoV-2 by directly binding to the N-terminal domain of the spike protein. Conversely, antihistamine drugs, primarily HRH1 antagonists, can competitively bind to HRH1 and thereby prevent viral entry. These findings revealed that the administration of repurposable antihistamine drugs could be a therapeutic intervention to combat coronavirus disease 19.
PubMed: 38953634
DOI: 10.1128/mbio.01088-24 -
MBio Jul 2024Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia...
Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus , BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has and properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.
PubMed: 38953633
DOI: 10.1128/mbio.00993-24 -
Journal of Virology Jul 2024Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1...
UNLABELLED
Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses.
IMPORTANCE
Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.
PubMed: 38953631
DOI: 10.1128/jvi.00499-24 -
General Physiology and Biophysics Jul 2024The work presents the connection between the infection of COVID-19 during pregnancy and non-syndromic orofacial clefts (NSOFC). Aim of the study was to compare the...
The work presents the connection between the infection of COVID-19 during pregnancy and non-syndromic orofacial clefts (NSOFC). Aim of the study was to compare the incidence of COVID-19 disease during mother´s pregnancy between a group of the children with NSOFC and a control group of the children without NSOFC. COVID-19 was confirmed by polymerase chain reaction (PCR) test. The study showed significantly higher incidence of COVID-19 disease in the group of mothers who gave birth to a child with NSOFC in comparison to the group of mothers who gave birth to a child without NSOFC. Our results indicate the possible participation of the infection of COVID-19 in the formation of NSOFCs.
Topics: Humans; Cleft Lip; Cleft Palate; COVID-19; Female; Pregnancy; Pregnancy Complications, Infectious; Incidence; Adult; Male; Infant, Newborn
PubMed: 38953573
DOI: 10.4149/gpb_2024012