The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. ...Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.
West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), ...saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.
West Nile virus (WNV) is an arbovirus with important public health implications globally. This study characterizes a viral isolate, 2004Hou3, in comparison with the NY99 strain from the original WNV ...outbreak in New York, USA. NextGen sequencing was used to compare the viral isolates genetically, while wild-type C57/BL6 mice were used to compare pathogenicity and viral persistence. Significant differences in survival and clinical presentations were noted, with minor genetic variations between the two strains potentially offering an explanation. One notable difference is that 5 of 35 mice infected with the 2004Hou3 strain developed hind limb flaccid paralysis, suggesting its possible use as a small animal pathogenesis model for this clinical characteristic often observed in human WN neuroinvasive disease patients but not reported in other animal models of infection. Overall, this study suggests that 2004Hou3 is a less pathogenic strain with potential for use in long-term outcome studies using small animal models.
G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome ...coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.
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•Structural and biochemical analyses define the basis of SARS-CoV-2 N interaction with G3BP1•F17A mutation of N protein specifically abolishes interaction with G3BP1/2•N-F17A attenuates SARS-CoV-2 replication and pathogenesis in vivo•G3BP1-N interaction suppresses G3BP1-dependent condensation of viral genomic RNA
Yang et al. characterize the interaction between G3BP1 and SARS-CoV-2 N protein at the single-residue level. They show that a point mutation (N-F17A) selectively disrupts G3BP1-N interaction and reduces viral replication and pathology in vivo, demonstrating that this interaction promotes infection by limiting sequestration of viral gRNA into stress granules.
Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds ...host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.
Synopsis
The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures.
The SARS-CoV-2 NSP3 protein binds to the KH domains of FMRPs through a short peptide motif.
Engineered SARS-CoV-2 viruses unable to bind FMRPs have reduced replication.
NSP3 binding to FMRPs disrupts their localization to stress granules through competition with UBAP2L.
The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures.
Summary
Incidence of peach Prunus persica (L.) Batsch tree mortality attributed to Armillaria root disease was assessed from 2009 to 2011 in 15 orchards in the State of Mexico, Mexico. Incidence ...increased gradually every year of assessment, reaching average values of 9.7, 15.3 and 20.3% tree mortality and 23.2, 24.7 and 28.3% disease‐impacted area of the orchards during 2009, 2010 and 2011, respectively. The cultivars ‘Nemaguard’ and ‘Criollo of La Goleta’, a local rootstock used in the region, were both susceptible to the disease. To identify species of Armillaria isolated from infected peach trees, two nuclear rDNA regions (partial 5.8S‐ITS2‐LSU D‐domains and partial 3′ LSU‐IGS1) and the translation elongation factor‐1α (tef‐1α) gene were sequenced and compared with sequences of known Armillaria species. DNA sequence analysis from 49 Armillaria isolates revealed that five isolates (10.2%) were Armillaria mellea and eight isolates (16.3%) were Armillaria gallica. DNA sequences from the remaining 36 isolates (73.5%) showed no close similarity to Armillaria sequences in GenBank, and apparently represent an undescribed Armillaria species. This undescribed species was the most widely distributed in the region of study. Separate phylogenetic analyses of the LSU region (D1–D3 domains concatenated with the partial 3′ end) and the tef‐1α region show that the undescribed species is quite distinct from other Armillaria spp. reported in North America.
We aimed to estimate the overall distribution of odontogenic infection by socio-demographic and medical characteristics in patients admitted to the Adult University Hospital (AUH) in Puerto Rico ...(PR).
A cross-sectional study was undertaken with the medical charts of 129 patients (≥21 years) with odontogenic infection who had been admitted (2011-2015) to the AUH and treated by the Oral and Maxillofacial Surgery Post - graduate Program of the University of PR. The patients were selected from the hospital's billing database after having been identified using the International Classification of Diseases (9th and 10th revisions). The study variables included age, gender, municipality of residence, medical insurance, infection etiology, surgical and antibiotic treatments, length of stay (LOS), and the presence of diabetes. Descriptive and frequency statistics were calculated for all the variables; chi-squared, Kruskal-Wallis, Kendall tau, and Mann-Whitney tests were performed. A P < .05 was considered statistically significant.
The mean age of the subjects was 40.36 (SD: 14.74) years, and they ranged in age from 21 to 81 years; the majority were enrolled in the public health insurance plan of PR. The leading cause of infection was dental caries. Diabetes was associated with longer LOSs; P < .01.
In our study, the relative frequency of admitted patients with an odontogenic infection, most of them with low income, increased over time with dental caries being the principal cause of infection.
In September 2007, rhizomorphs with morphological characteristics of Armillaria were collected from woody hosts in forests of Mexico State, Veracruz, and Oaxaca, Mexico. Based on pairing tests, ...isolates were assigned to five somatically compatible genets or clones (MEX7R, MEX11R, MEX23R, MEX28R, and MEX30R). These genets were all identified as Armillaria gallica based on somatic pairing tests against known tester isolates and nucleotide sequences of the translation elongation factor 1α (tef-1α; GenBank Accession Nos. KF156772 to 76). Sequences of tef-1α for all genets showed a max identity of 97 to 99% to A. gallica (ST23, JF313125) (3,4). However, A. gallica comprises a genetically diverse complex that likely represents multiple cryptic species (3). In Mexico, this species has been previously reported in northeastern Morelos on Quercus sp., eastern Mexico State on Pinus hartwegii, and southwestern Mexico State on Prunus persica (1,2). This study identified associations with 10 new hosts within three states of Mexico, but only five hosts were diseased. Genet MEX7R comprised seven isolates collected in the University of Chapingo forest near Texcoco, Mexico State (19°18'10.764″ N, 98°42'14.147″ W, elevation 3441 m). Four MEX7R isolates were collected from diseased Alnus sp. including the root ball of a 130 cm dbh, root-disease killed tree, one isolate from a symptomless Senecio sp. s.l. (Roldana sp.) shrub and two isolates from symptomless Abies religiosa. Genet MEX11R comprised four isolates from a cloud forest near Xalapa, Veracruz (19°31'14.628″ N, 96°59'22.812″ W, elevation 1496 m). MEX11R isolates were collected from the roots of a root-disease killed Carpinus caroliniana, and from trees with no obvious symptoms (Miconia mexicana, Quercus xalapensis, and Liquidambar styraciflua). Two isolates of genet MEX23R were collected from the Jardin Botanico Francisco Javier Clavijero, Instituto de Ecologia, A.C., Xalapa, Veracruz (19°30'49.067″ N, 96°56'32.999″ W, elevation 1344 m). These isolates were from root-diseased Eriobotrya japonica (non-native fruit tree) that showed obvious symptoms (flaccid, chlorotic, and senescing leaves) and from an adjacent, infected Platanus mexicana that did not show readily observable symptoms. Two collections near Oaxaca, Oaxaca, included a single isolate MEX28R from the root ball of a recently root disease-killed Arbutus xalapensis within a small root disease center at Peña Prieta, in Parque La Cumbre, near Ixtepeji (17°09'42.084″ N, 96°38'15.936″ W, elevation 2853 m) and a single isolate MEX30R from the base of an asymptomatic Alnus acuminata near the El Carrizal fish hatchery 10 km northeast of San Miguel del Valle (17°06'45.036″ N, 96°24'03.743″ W, elevation 2594 m). Armillaria gallica has a circumpolar distribution with an extremely wide host range, and its ecological behavior varies greatly. Continued surveys are needed to better understand the distribution and ecological impacts of this pathogen in relation to Armillaria root disease in Mexico and the potential influences of climate change. Although A. gallica displays diverse ecological behavior, trees infected with A. gallica are less likely to survive the stresses of human activity and a changing climate (4). References: (1) D. Alvarado-Rosales and R. A. Blanchette. Phytopathology 84:1106, 1994. (2) R. D. Elias-Roman et al. For. Pathol. 43:390, 2013. (3) M.-S. Kim et al. Phytopathology 102:S4.63, 2012. (4) B. Marcais and N. Breda. J. Ecol. 94:1214, 2006.
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