Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma.

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies.

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.

Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis.

Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

Effect of yeast (Xanthophyllomyces dendrorhous) and plant (Saint John's wort, lemon balm, and rosemary) extract based functional diets on antioxidant and immune status of Atlantic salmon (Salmo salar) subjected to crowding stress.

Salmon farming may face stress due to the intensive culture conditions with negative impacts on overall performance. In this aspect, functional feed improves not only the basic nutritional requirements but also the health status and fish growth. However, to date no studies have been carried out to evaluate the effect of functional diets in salmon subjected to crowding stress. Thus, the aim of this study was to evaluate the effect of yeast extract (Xanthophyllomyces dendrorhous; diet A) and the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary; diet B) on the antioxidant and immune status of Atlantic salmon grown under normal cultured conditions and then subjected to crowding stress. Fish were fed with functional diets during 30 days (12 kg/m3) and then subjected to crowding stress (20 kg/m3) for 10 days. The lipid peroxidation in gut showed that both diets induced a marked decrease on oxidative damage when fish were subjected to crowding stress. The protein carbonylation in muscle displayed at day 30 a marked decrease in both functional diets that was more marked on the stress condition. The expression of immune markers (IFNγ, CD4, IL-10, TGF-ß, IgMmb, IgMsec, T-Bet, and GATA-3) indicated the upregulation of those associated to humoral-like response (CD4, IL-10, GATA-3) when fish were subjected to crowding stress. These results were confirmed with the expression of secreted IgM. Altogether, these functional diets improved the antioxidant status and increased the expression of genes related to Th2-like response suggesting a protective role on fish subjected to crowding stress.

Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant.

Adjuvants used in vaccine aquaculture are frequently harmful for the fish, causing melanosis, granulomas and kidney damage. Along with that, vaccines are mostly administered by injection, causing pain and stress to the fish. We used the DNA coding for the replicase of alphavirus as adjuvant (Ad) of a vaccine against ISAV. The Ad and an inactivated ISAV (V) were loaded in chitosan nanoparticles (NPs) to be administered orally to Atlantic salmon. NP-Ad was able to deliver the DNA ex vivo and in vivo. Oral administration of the NPs stimulated the expression of immune molecules, but did not stimulate the humoral response. Although the vaccination with NP-V results in a modest protection of fish against ISAV, NP-V administered together with NP-Ad caused a protection of 77%. Therefore, the DNA coding for the replicase of alphavirus could be administered orally and can potentiate the immuneprotection of a virine against infection.

Development of a reverse genetic system for infectious salmon anemia virus: rescue of recombinant fluorescent virus by using salmon internal transcribed spacer region 1 as a novel promoter.

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV(901_09) and rISAVr(S6-NotI-HPR) containing a NotI restriction site and rISAV(S6/EGFP-HPR) harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5105 PFU/ml,similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.

Genomic adaptation of the ISA virus to Salmo salar codon usage.

BACKGROUND: The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. METHODS: Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. RESULTS: Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. CONCLUSIONS: Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations.

Analysis of the use of codon pairs in the HE gene of the ISA virus shows a correlation between bias in HPR codon-pair use and mortality rates caused by the virus.

BACKGROUND: Segment 6 of the ISA virus codes for hemoagglutinin-esterase (HE). This segment is highly variable, with more than 26 variants identified. The major variation is observed in what is called the high polymorphism region (HPR). The role of the different HPR zones in the viral cycle or evolution remains unknown. However viruses that present the HPR0 are avirulent, while viruses with important deletions in this region have been responsible for outbreaks with high mortality rates. In this work, using bioinformatic tools, we examined the influence of different HPRs on the adaptation of HE genes to the host translational machinery and the relationship to observed virulence. METHODS: Translational efficiency of HE genes and their HPR were estimated analyzing codon-pair bias (CPB), adaptation to host codon use (codon adaptation index-CAI) and the adaptation to available tRNAs (tAI). These values were correlated with reported mortality for the respective ISA virus and the ΔG of RNA folding. tRNA abundance was inferred from tRNA gene numbers identified in the Salmo salar genome using tRNAScan-SE. Statistical correlation between data was performed using a non-parametric test. RESULTS: We found that HPR0 contains zones with codon pairs of low frequency and low availability of tRNA with respect to salmon codon-pair usage, suggesting that HPR modifies HE translational efficiency. Although calculating tAI was impossible because one third of tRNAs (~60.000) were tRNA-ala, translational efficiency measured by CPB shows that as HPR size increases, the CPB value of the HE gene decreases (P = 2x10⁻7, ρ = -0.675, n = 63) and that these values correlate positively with the mortality rates caused by the virus (ρ = 0.829, P = 2x10⁻7, n = 11). The mortality associated with different virus isolates or their corresponding HPR sizes were not related with the ΔG of HPR RNA folding, suggesting that the secondary structure of HPR RNA does not modify virulence. CONCLUSIONS: Our results suggest that HPR size affects the efficiency of gene translation, which modulates the virulence of the virus by a mechanism similar to that observed in production of live attenuated vaccines through deoptimization of codon-pair usage.

Nanoparticles and microparticles of polymers and polysaccharides to administer fish vaccines.

Aquaculture has become an important economic sector worldwide, but is faced with an ongoing threat from infectious diseases. Vaccination plays a critical role in protecting commercially raised fish from bacterial, viral and parasitic diseases. However, the production of effective vaccines is limited by the scarcity of knowledge about the immune system of fish. Improving vaccines implies using antigens, adjuvants and employing methods of administration that are more effective and less harmful to the fish. In this context, in recent year there have studies of methods of encapsulating antigens in matrices of different types to apply in fish vaccines. This work reviews the new methods to improve fish vaccines by encapsulating them in polymers and polysaccharides.

Nanoparticles and microparticles of polymers and polysaccharides to administer fish vaccines

Aquaculture has become an important economic sector worldwide, but is faced with an ongoing threat from infectious diseases. Vaccination plays a critical role in protecting commercially raised fish from bacterial, viral and parasitic diseases. However, the production of effective vaccines is limited by the scarcity of knowledge about the immune system of fish. Improving vaccines implies using antigens, adjuvants and employing methods of administration that are more effective and less harmful to the fish. In this context, in recent year there have studies of methods of encapsulating antigens in matrices of different types to apply in fish vaccines. This work reviews the new methods to improve fish vaccines by encapsulating them in polymers and polysaccharides.

Antiviral activity in vitro and in vivo of natural flavonoids isolated from Heliotropium sinuatum against infectious salmon anemia virus (ISAV) / Actividad antiviral in vitro e in vivo de flavonoides naturales aislados desde Heliotropium sinuatum contra el virus de la anemia infecciosa en salmones (ISAV)

The infectious salmon anemia virus (ISAV) of Orthomyxoviridae family, is responsible for heavy losses in industry aquaculture around the world, affecting several commercial aquatic organisms, mainly Salmo salar. Therefore, it is important to find effective antiviral therapies. In this work we evaluated in vitro and in vivo the antiviral activity of three natural flavonoids isolated from the resinous exudates of the plant Heliotropium sinuatum (Heliotropiaceae) against ISAV. The results show that 7-O-methyleriodictyol was able to inhibit the infectivity of ISAV in vitro assay with EC 50 of 0.20 ug/mL. Despite having a citotoxicity expressed as CC50 of 12.80 ug/mL, the in vivo study showed that this compound protected 100 percent to the fish infected with ISAV keeping 100 percent fish viability. These results allow the proposal of 7-O-methyleriodictyol as a good candidate to be used as antiviral therapy for ISAV in salmon industry.

El virus de la anemia infecciosa en salmón de la familia Orthomyxoviridae, es el responsable de grandes pérdidas en la industria acuícola alrededor del mundo, afectando diversas especies acuáticas comerciales, principalmente Salmo salar. Por lo tanto, es muy importante encontrar una terapia antiviral efectiva. En el presente trabajo, evaluamos la actividad antiviral in vitro e in vivo de tres flavonoides naturales aislados desde el exudado resinoso de la especie vegetal Heliotropium sinuatum (Heliotropiaceae) contra ISAV. Los resultados mostraron que 7-O-metileriodictiol inhibió la infectividad de ISAV in vitro con un EC50 de 0.20 ug/mL. A pesar de tener una citotoxicidad expresada como un CC50 de 12.80 ug/mL, el estudio in vivo mostró que este compuesto protege en un 100 por ciento a los peces infectados con ISAV manteniendo un 100 por ciento de viabilidad. Estos resultados permiten proponer que 7-O-metileriodictiol es un buen candidato para ser usado como terapia antiviral para ISAV en la industria salmonera.

Inhibitory effect of a nucleotide analog on infectious salmon anemia virus infection.

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-ß-d-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC50) of 0.02 µg/ml and an IC90 of 0.4 µg/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo.

Infectious salmon anemia virus--genetics and pathogenesis.

The infectious salmon anemia virus (ISAV) is the causative agent of the ISA syndrome that affects mainly Atlantic salmon (Salmo salar) and has caused high mortality epidemics in Norway, Scotland, Canada, the United States and Chile. It is classified as an Orthomyxoviridae, its genome is composed of 8 single-strand RNA segments with negative polarity that code for 11 polypeptides. Through functional studies of the coded proteins it has been established that RNA segments 5 and 6 code for a fusion protein and hemagglutinin, respectively, while two polypeptides coded by segments 7 and 8 inhibit interferon induction. The functions of the rest of the possible proteins coded by the viral genome have been assigned by comparison with the corresponding ones of the influenza virus genome. As to its pathogenicity, some growth parameters such as incubation period, resistance to chemical and physical factors, establishment of the infection in other marine species, and dissemination ability among the different organs have been evaluated in several salmonids. Genomic analysis has shown (i) the existence of a high polymorphism region (HPR) in segment 6, and (ii) sequence insertion in segment 5. More than 20 HPR variants have been determined, all originating from HPR0, which is associated with low pathogenicity, while 4 different sequence insertions in segment 5 have not been related with some characteristic of the virus infection. Much progress has been made in the characterization of the virus in 20 years of study, but more detailed knowledge of the specific function of the proteins coded by all the viral genes is still missing, including the pathogenicity mechanism at the molecular level.

An ATPase activity associated with the rotavirus phosphoprotein NSP5.

Interactions between NSP5 and NSP2 drive the formation of viroplasms, sites of genome replication and packaging in rotavirus-infected cells. The serine-threonine-rich NSP5 transitions between hypo- and hyper-phosphorylated isomers during the replication cycle. In this study, we determined that purified recombinant NSP5 has a Mg2+-dependent ATP-specific triphosphatase activity that generates free ADP and Pi (Vmax of 19.33 fmol of product/min/pmol of enzyme). The ATPase activity was correlated with low levels of NSP5 phosphorylation, suggestive of a possible link between ATP hydrolysis and an NSP5 autokinase activity. Mutagenesis showed that the critical residue (Ser67) needed for NSP5 hyperphosphorylation by cellular casein kinase-like enzymes has no role in the ATPase or autokinase activities of NSP5. Through its NDP kinase activity, the NSP2 octamer may support NSP5 phosphorylation by creating a constant source of ATP molecules for the autokinase activity of NSP5 and for cellular kinases associated with NSP5.

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein.

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.

[Chronicle of an announced pandemic?]. / Crónica de una pandemia anunciada?

In the last years our country has been affected by several outbreaks of infectious diseases such as Cholera and Hanta virus and recently, by pathogens associated to red tide. Chile was able to manage those emergencies using the local health system. The new threat that may emerge and could eventually overcome that capacity, is the possible H5N1 influenza virus outbreak. Influenza is responsible for the most destructive pandemic, the Spanish influenza, that killed over 40 million individuals in 1918. The new influenza strain (H5N1) is at present endemic in poultry in Asia and has been associated to human fatal cases in Hong Kong and Vietnam. Even though this strain is not able yet to be transmitted among humans, evidence has accumulated that such ability could be reached by the new strain, since it was already detected in pigs. That particular evidence may indicate that the virus could adapt to infect humans, since a similar situation was observed in several of the influenza pandemics. The World Health Organization set a "task force" to develop a strategy that may help to control the virus spread. Several countries are already stocking anti-flu drugs and others are developing new vaccine that are currently been assayed in human volunteers. It is possible that we may have a vaccine before the outbreak; this development is even faster than for SARS. The mayor question to be addressed for developing countries is: what will be done if we do not have the vaccine on time?

Replication and transcription of the rotavirus genome.

Rotaviruses are an important cause of human morbidity and mortality, representing the primary pathogens responsible for acute dehydrating diarrhea in children under the age of 3. The infectious rotavirus particle is made up of three concentric layers of protein, and contains a genome consisting of eleven segments of double-stranded (ds)RNA. Upon infection, RNA polymerases associated with double-layered virus particles are activated, resulting in genome transcription and extrusion of the eleven viral mRNAs from such particles. The mRNAs not only direct protein synthesis, but also serve as templates for minus-strand synthesis to yield dsRNAs. Synthesis of the dsRNAs is an event that occurs following the gene-specific packaging of viral mRNAs into core-like assembly intermediates. Electron-dense cytoplasmic inclusions, termed viroplasms, function as sites of genome packaging and replication in the infected cell. Our understanding of key events in the viral life cycle has been advanced considerably by the development of cell-free systems that support mRNA synthesis from virion-derived double-layered particles and dsRNA synthesis from virion-derived core particles. The recent expression and purification of rotavirus recombinant proteins have also allowed progress to be made in defining the roles of viral proteins in genome replication and viroplasm formation. However, our efforts towards a full description of the viral life cycle, most notably an understanding of the events occurring during gene-specific packaging, remain hampered by the lack of a cell-free packaging system and a reverse genetics systems. The lack of a reverse genetics systems also confounds efforts towards the generation of molecular engineered second-generation vaccines.

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2.

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (HØHØHØØ where Ø is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.