Roles of VP4 and NSP1 in determining the distinctive replication capacities of simian rotavirus RRV and bovine rotavirus UK in the mouse biliary tract

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1.     

Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.     

Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence.

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.     

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-α/β and IFN-γ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-α/β receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-γ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.     

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.     

Computational identification of post-translational modification sites and functional families reveal possible moonlighting role of rotaviral proteins

Rotavirus (RV) diarrhoea causes huge number deaths in children less than 5 years of age. In spite of available vaccines, it has been difficult to combat RV due to large number of antigenically distinct genotypes, high mutation rates, generation of reassortant viruses due to segmented genome. RV is an eukaryotic virus which utilizes host cell machinery for its propagation. Since RV only encodes 12 proteins, post-translational modification (PTM) is important mechanism for modification, which consequently alters their function. A single protein exhibiting different functions in different locations or in different subcellular sites, are known to be 'moonlighting'. So there is a possibility that viral proteins moonlight in separate location and in different time to exhibit diverse cellular effects. Based on the primary sequence, the putative behaviour of proteins in cellular environment can be predicted, which helps to classify them into different functional families with high reliability score. In this study, sites for phosphorylation, glycosylation and SUMOylation of the six RV structural proteins (VP1, VP2, VP3, VP4, VP6 & VP7) & five non-structural proteins (NSP1, NSP2,NSP3,NSP4 & NSP5) and the functional families were predicted. As NSP6 is a very small protein and not required for virus growth & replication, it was not included in the study. Classification of RV proteins revealed multiple putative functions of each structural protein and varied number of PTM sites, indicating that RV proteins may also moonlight depending on requirements during viral life cycle. Targeting the crucial PTM sites on RV structural proteins may have implications in developing future anti-rotaviral strategies.     

Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo.

To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.     

Feeling manipulated: cytomegalovirus immune manipulation

Background

ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication.

Results

We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses.

Conclusion

The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.     

G5型人A组轮状病毒LL36755株的VP4、VP6和NSP4编码基因的分子特征

最近在亚洲首次发现并报道了感染人的G5型人A组轮状病毒LL36755株,为进一步探讨其进化来源,克隆了G5型人A组轮状病毒LL36755株的VP4、VP6、NSP4编码基因,并分析其基因序列的分子特征。结果发现卢龙株LL36755为罕见的G5P[6]型,其VP6的亚群为SGⅡ型,NSP4的基因型为B型。系统进化树分析表明,卢龙株LL36755的VP7、VP4编码基因与猪来源的毒株关系密切,而VP6、NSP4编码基因与人来源的毒株紧密相联系。可以推断新的人腹泻A组轮状病毒LL36755株是猪的VP7,VP4编码基因与人的VP6,NSP4编码基因的自然重组;而且该毒株不是G5的原型,很可能是人类轮状病毒与猪轮状病毒毒株的自然重组后逐步进化而来。     

Proteomic methods reveal cyclophilin a function as a host restriction factor against rotavirus infection

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl‐prolyl cis‐trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.     

A lymphatic mechanism of rotavirus extraintestinal spread in the neonatal mouse

We used the neonatal mouse model of rotavirus infection and virus strains SA11-clone 4 (SA11-Cl4) and Rhesus rotavirus (RRV) to examine the mechanism of the extraintestinal spread of viruses following oral inoculation. The spread-competent viruses, RRV and reassortant R7, demonstrated a temporal progression from the intestine, to the terminal ileum, to the mesenteric lymph nodes (MLN), and to the peripheral tissues. SA11-Cl4 was not found outside the intestine. Reassortant virus S7, which was unable to reach the liver in previous studies (E. C. Mossel and R. F. Ramig, J. Virol. 76:6502-6509, 2002), was recovered from 60% of the MLN, suggesting that there are multiple determinants for the spread of virus from the intestine to the MLN. Phenotypic segregation analysis identified RRV genome segment 6 (VP6) as a secondary determinant of the spread of virus to the MLN (P = 0.02) in reassortant viruses containing segment 7 from the spread-incompetent parent. These data suggest that in the orally infected neonatal mouse, the extraintestinal spread of rotavirus occurs via a lymphatic pathway, and the spread phenotype is primarily determined by NSP3 and can be modified by VP6.     

Characterization of homologous and heterologous rotavirus-specific T-cell responses in infant and adult mice

During primary rotavirus (RV) infection, CD8+ T cells play an important role in viral clearance as well as providing partial protection against reinfection. CD4+ T cells are essential for maximal development of RV-specific intestinal immunoglobulin A. In this study, we took advantage of the cytokine flow cytometry technique to obtain a detailed map of H-2b- and H-2d-restricted CD8+ and CD4+ T-cell epitopes from the RV proteins VP6 and VP7. Three new CD8+ T-cell epitopes (H-2d and H-2b restricted) and one new CD4+ T-cell epitope (H-2d and H-2b restricted) were identified. Using these newly identified targets, we characterized the development and specificity of cellular immune responses in C57BL/6 and BALB/c mice during acute infection of infants and adults. We found that both the CD4+ and CD8+ responses peaked on days 5 to 7 after infection and then declined rapidly. Interestingly, both the response kinetics and tissue distributions were different when epitopes on VP6 and VP7 were compared. VP6 elicited a response which predominated in the intestine, while the response to VP7 was more systemic. Additionally, the T-cell responses elicited after homologous versus heterologous infection differed substantially. We found that during homologous infection, there was a greater response toward VP6 than that toward VP7, especially in the intestine, while after heterologous infection, this was not the case. Finally, in suckling mice, we found two peaks in the CD8 response on days 7 and 14 postinfection, which differed from the single peak found in adults and likely mimics the biphasic pattern of rotavirus shedding in infant mice.     

Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes

Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca(2+) concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.     

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.