SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.

YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 small ubiquitin-like modifier (SUMO) ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 synergizes with wild-type YTHDF2, but not a SUMOylation-deficient mutant, to limit EBV lytic replication. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3, to restrict EBV replication. These results collectively uncover a unique mechanism whereby YTHDF family proteins control EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.IMPORTANCEm6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.

SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.

YTHDF2 is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 SUMO ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 enhances the antiviral activity of YTHDF2, and SUMOylation-deficient YTHDF2 shows reduced anti-EBV activity. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3. These results collectively uncover a unique mechanism whereby YTHDF2 controls EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.

PIAS1 potentiates the anti-EBV activity of SAMHD1 through SUMOylation.

BACKGROUND: Sterile alpha motif and HD domain 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase (dNTPase) that restricts the infection of a variety of RNA and DNA viruses, including herpesviruses. The anti-viral function of SAMHD1 is associated with its dNTPase activity, which is regulated by several post-translational modifications, including phosphorylation, acetylation and ubiquitination. Our recent studies also demonstrated that the E3 SUMO ligase PIAS1 functions as an Epstein-Barr virus (EBV) restriction factor. However, whether SAMHD1 is regulated by PIAS1 to restrict EBV replication remains unknown. RESULTS: In this study, we showed that PIAS1 interacts with SAMHD1 and promotes its SUMOylation. We identified three lysine residues (K469, K595 and K622) located on the surface of SAMHD1 as the major SUMOylation sites. We demonstrated that phosphorylated SAMHD1 can be SUMOylated by PIAS1 and SUMOylated SAMHD1 can also be phosphorylated by viral protein kinases. We showed that SUMOylation-deficient SAMHD1 loses its anti-EBV activity. Furthermore, we demonstrated that SAMHD1 is associated with EBV genome in a PIAS1-dependent manner. CONCLUSION: Our study reveals that PIAS1 synergizes with SAMHD1 to inhibit EBV lytic replication through protein-protein interaction and SUMOylation.

Intestinal Tropism of an Infectious Bronchitis Virus Isolate Not Explained by Spike Protein Binding Specificity.

An infectious bronchitis virus (IBV) with an unusual enteric tropism (CalEnt) was isolated from a California broiler flock exhibiting runting-stunting syndrome. IBV was detected in the small intestine, but not in the respiratory tract or kidney. During virus isolation in embryos, it did not replicate in chorioallantoic membrane (CAM) but could be recovered from intestines. Its S1 protein showed 93% amino acid sequence identity to a California variant isolated in 1999 (Cal99). Intestinal lesions were reproduced following ocular/nasal inoculation of specific-pathogen-free chickens, but respiratory signs and lesions were also present. The virus was detected in both respiratory and intestinal tissues. To determine whether the novel tropism of IBV CalEnt was due to an increased ability of its S1 protein to bind to the intestinal epithelium, we compared the binding of soluble trimeric recombinant S1 proteins derived from CalEnt and Cal99 to chicken tissues. Contrary to expectations, the CalEnt S1 protein did not bind to small intestine and, unlike Cal99 S1, did not bind to the respiratory epithelium or CAM. Using only the CalEnt S1 N-terminal domain or including the S2 ectodomain (lacking membrane and cytoplasmic domains), which have been shown to improve ArkDPI S1 protein binding, did not lead to detectable binding at the standard protein concentration to any tissue tested. Our results indicate no/poor binding of the CalEnt spike protein to both respiratory and intestinal tissues and thus do not support better attachment to intestinal epithelial cells as a reason for CalEnt's extended tropism. These results might reflect shortcomings of the assay, including that it does not detect potential contributions of the S1 C-terminal domain to attachment. We used bioinformatic approaches to explore the possibility that the unique tropism of CalEnt might be a result of functions of the S protein in cell-entry steps subsequent to attachment. These analyses suggest that CalEnt's S2 coding region was acquired through a recombination event and encodes a unique amino acid sequence at the putative recognition site for the protease that activates the S protein for fusion. Thus, S2 activation by tissue-specific proteases might facilitate CalEnt entry into intestinal epithelial cells and compensate for poor binding by its S1 protein.

Tropismo intestinal de un aislamiento del virus de la bronquitis infecciosa con una especificidad de unión a la proteína espícula inusual. Se aisló un virus de la bronquitis infecciosa (IBV) con un tropismo entérico inusual (CalEnt) de una parvada de pollos de engorde de California que presentaba síndrome de retraso en el crecimiento. Se detectó al virus de bronquitis en el intestino delgado, pero no en el tracto respiratorio o en el riñón. Durante el aislamiento del virus en huevos embrionados de pollo, no se replicó en la membrana corioalantoidea (CAM), pero pudo recuperarse de los intestinos. Su proteína S1 mostró una identidad de secuencia de aminoácidos del 93% con una variante de California aislada en el año 1999 (Cal99). Las lesiones intestinales se reprodujeron después de la inoculación ocular/nasal de pollos libres de patógenos específicos, pero también hubo signos y lesiones respiratorias. El virus se detectó en los tejidos respiratorios e intestinales. Para determinar si el nuevo tropismo de este virus de la bronquitis infecciosa CalEnt se ocasionaba por una mayor capacidad de su proteína S1 para unirse al epitelio intestinal, se comparó la unión a los tejidos de pollo de las proteínas S1 recombinantes triméricas solubles derivadas de los virus CalEnt y Cal99. Contrariamente a lo esperado, la proteína CalEnt S1 no se unió al intestino delgado y a diferencia del virus Cal99 S1, no se unió al epitelio respiratorio o CAM. Mediante el uso de solo el dominio N-terminal de la proteína S1 del virus CalEnt o por la inclusión del ectodominio S2 (que carece de dominios de membrana y citoplasmáticos), que se ha demostrado mejora la unión de la proteína S1 del serotipo Arkansas DPI, no se observó una unión detectable a ningún tejido analizado a la concentración de proteína estándar. Estos resultados indican una unión nula o deficiente de la proteína de la espícula del virus CalEnt a los tejidos respiratorios e intestinales y por lo tanto, no respaldan la preferencia de la unión a las células epiteliales intestinales como una razón para el tropismo extendido del virus CalEnt. Estos resultados pueden reflejar las deficiencias del ensayo, incluyendo el hecho de que no detecta posibles contribuciones del dominio C-terminal de la proteína S1 en la unión. Se utilizaron enfoques bioinformáticos para explorar la posibilidad de que el tropismo único del virus CalEnt podría ser el resultado de las funciones de la proteína S en los pasos de entrada a las células posteriores a la unión. Estos análisis sugieren que la región de codificación S2 del virus CalEnt se adquirió a través de un evento de recombinación y codifica una secuencia de aminoácidos única en el supuesto sitio de reconocimiento de la proteasa que activa la proteína S para la fusión. Por lo tanto, la activación de S2 por proteasas específicas de tejido podría facilitar la entrada del virus CalEnt en las células epiteliales intestinales y compensar la unión deficiente por su proteína S1.

Co-circulation of G1, G2 and G9 rotaviruses in hospitalized patients in Bangladesh during 2006-2009.

Rotavirus strain diversity in Bangladesh has been explored since 1985 and as seen in other parts of the world, rotaviruses have shown tremendous strain diversity overtime. Rotavirus antigen was detected in stool specimens using a solid-phase sandwich-type enzyme immunoassay. A multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for rotavirus G and P genotypes. This current study was carried out between 2006 and 2009 during which time 1,607 (23%) of 7,058 fecal specimens tested positive for group A rotaviruses with the highest incidence rate being observed in winter each year. Genotyping of rotaviruses showed a sharp decline in G2P[4] between 2008 and 2009 with a gradual increase in G1 and G9 strains. Since the Government of Bangladesh is planning to include rotavirus vaccine in the national immunization program, these data on rotavirus strain diversity should be taken into consideration for vaccine strain selection.

Clinical presentation and molecular characterization of group B rotaviruses in diarrhoea patients in Bangladesh.

A total of 1106 stool samples collected from diarrhoea patients admitted to Dhaka hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, during January-December 2008 were analysed for the presence of rotavirus-specific RNA by PAGE. The group B-specific RNA migration pattern was detected in 26 patients (2.4%) and group A-specific pattern in 259 patients (23.4%). Clinical data from group A and group B rotavirus-infected patients indicated that episodes did not differ much in the prevalence of diarrhoea, number of stools, outcome or differences in gender. However, abdominal pain was more common in group B rotavirus infections (36 vs 15%, P=0.02) and the virus was responsible for more severe dehydration compared with group A-infected patients (12 vs 3%, P=0.04). Sequence analyses of VP4, VP7 and NSP2 indicated that an Indian-Bangladeshi lineage of the virus, which is different from both the prototype (Chinese) lineage and from the animal group B rotaviruses, has been circulating in Bangladesh. Continuous monitoring of group B rotaviruses both in hospitals and in the community will be helpful to determine the true burden of group B rotaviruses.

Complete genomic analysis of a Bangladeshi G1P[8] rotavirus strain detected in 2003 reveals a close evolutionary relationship with contemporary human Wa-like strains.

More than 120 variants of rotavirus strains with different VP7 (G type) and VP4 (P type) combinations are reported thus far. Among them Wa-like G1P[8] rotaviruses are the most common human strains worldwide. However, characterization of their entire genome complement is limited to a few old prototype strains, and no complete genome data for any G1P[8] strain isolated in the last decade are available. Both the currently licensed rotavirus vaccines Rotarix and RotaTeq possess the G1 and P[8] specificities. Therefore, comprehensive genetic information of the currently circulating G1P[8] strain is important to assess the impact of rotavirus vaccines on the circulating rotavirus strains. Here we report the complete genome sequence of a G1P[8] rotavirus strain Dhaka16-03 isolated in 2003 from a Bangladeshi child hospitalized with severe diarrhea. Based on a full-genome classification system, Dhaka16-03 was shown to posses the typical Wa-like genotype constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1. The strain was phylogenetically more closely related to contemporary human rotavirus strains (isolated in the 2000s) with a range of G and P-genotypes than to those of the prototype G1P[8] strains. Since the vaccine strains are developed based on strains isolated several decades ago, it is important to know how much the vaccine strains differ from the currently circulating G1P[8] and other Wa-like strains. Our complete genome characterization of a recent G1P[8] strain will be helpful to assess the ongoing rotavirus vaccine trials and their implementation programs in the forthcoming years.

Prevalence of G2P[4] and G12P[6] rotavirus, Bangladesh.

Approximately 20,000 stool specimens from patients with diarrhea visiting 1 urban and 1 rural hospital in Bangladesh during January 2001-May 2006 were tested for group A rotavirus antigen, and 4,712 (24.0%) were positive. G and P genotyping was performed on a subset of 10% of the positive samples (n = 471). During the 2001-2005 rotavirus seasons, G1P[8] (36.4%) and G9P[8] (27.7%) were the dominant strains, but G2[4] and G12P[6] were present in 15.4% and 3.1% of the rotavirus-positive patients, respectively. During the 2005-06 rotavirus season, G2P[4] (43.2%) appeared as the most prevalent strain, and G12P[6] became a more prevalent strain (11.1%) during this season. Because recently licensed rotavirus vaccines include only the P[8] specificity, it is unknown how the vaccines will perform in settings where non-P[8] types are prevalent.

Sequence analysis and evolution of group B rotaviruses.

Human group B rotaviruses were isolated from hospitalized patients in Bangladesh between July 2003 and December 2004. Phylogenetic analyses of the gene segments encoding the hemagglutinin (VP4), glycoprotein (VP7) and RNA-binding protein (NSP2) of group B rotaviruses showed that Bangladeshi strains were more similar to the Indian strains than to the prototype Chinese strains. Moreover, all human strains were clustered together and were distantly related to the animal strains. With limited sequence data, the evolutionary rate of the glycoproteins (VP7) of human group B rotaviruses was estimated to be 1.57x10(-3) nucleotide substitutions/(siteyear), which was comparable to other rapidly evolving RNA viruses. The most recent common ancestor (MRCA) of the extant human group B rotaviruses was calculated to date to around 1976.