Influenza Virus in a Natural Host,the Mallard: Experimental Infection Data

Wild waterfowl, particularly dabbling ducks such as mallards (Anas platyrhynchos), are considered the main reservoir of low-pathogenic avian influenza viruses (LPAIVs). They carry viruses that may evolve and become highly pathogenic for poultry or zoonotic. Understanding the ecology of LPAIVs in these natural hosts is therefore essential. We assessed the clinical response, viral shedding and antibody production of juvenile mallards after intra-esophageal inoculation of two LPAIV subtypes previously isolated from wild congeners. Six ducks, equipped with data loggers that continually monitored body temperature, heart rate and activity, were successively inoculated with an H7N7 LPAI isolate (day 0), the same H7N7 isolate again (day 21) and an H5N2 LPAI isolate (day 35). After the first H7N7 inoculation, the ducks remained alert with no modification of heart rate or activity. However, body temperature transiently increased in four individuals, suggesting that LPAIV strains may have minor clinical effects on their natural hosts. The excretion patterns observed after both re-inoculations differed strongly from those observed after the primary H7N7 inoculation, suggesting that not only homosubtypic but also heterosubtypic immunity exist. Our study suggests that LPAI infection has minor clinically measurable effects on mallards and that mallard ducks are able to mount immunological responses protective against heterologous infections. Because the transmission dynamics of LPAIVs in wild populations is greatly influenced by individual susceptibility and herd immunity, these findings are of high importance. Our study also shows the relevance of using telemetry to monitor disease in animals.     

How Host Population Dynamics Translate into Time-Lagged Prevalence: An Investigation of Sin Nombre Virus in Deer Mice

Human cases of hantavirus pulmonary syndrome caused by Sin Nombre virus are the endpoint of complex ecological cascade from weather conditions, population dynamics of deer mice, to prevalence of SNV in deer mice. Using population trajectories from the literature and mathematical modeling, we analyze the time lag between deer mouse population peaks and peaks in SNV antibody prevalence in deer mice. Because the virus is not transmitted vertically, rapid population growth can lead initially to reduced prevalence, but the resulting higher population size may later increase contact rates and generate increased prevalence. Incorporating these factors, the predicted time lag ranges from 0 to 18 months, and takes on larger values when host population size varies with a longer period or higher amplitude, when mean prevalence is low and when transmission is frequency-dependent. Population size variation due to variation in birth rates rather than death rates also increases the lag. Predicting future human outbreaks of hantavirus pulmonary syndrome may require taking these effects into account.     

Experimental Myocarditis Induced in Mice by Infection with Influenza A2 Virus

A mouse model was established for the study of acute myocarditis that occurs during influenza infection. Challenge with more than 10 LD₅₀ of mouse-adapted influenza A₂ virus (H₂N₂) induced myocarditis macroscopically discernible as white, irregularly shaped lesions which were shown by histological examination to consist of necrotic myofibers surrounded by infiltrating mononuclear inflammatory cells. After challenge with 10 LD₅₀ of the virus, macroscopic myocarditis was found to advance in a progressive manner up to the 7th day, while the virus titer in the heart reached its peak on the 2nd day and began to decrease on the 5th day of infection. However, development of myocarditis was significantly suppressed in mice which were irradiated with 400 R of X-rays before infection. In addition, myocarditis did not develop in congenitally athymic nude mice. These data indicate that myocarditis was not brought about by viral action directly, but that it was mediated by some function of the host against viral in-vasion, which was abolished by X-irradiation. The data also suggest that T cells played a key role in the development of myocarditis.     

Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD₅₀) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.The recent outbreaks of H5N1 avian flu and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV) are clear indications of the urgent need for effective vaccines against influenza A viruses (31). Preventive and therapeutic measures against influenza A viruses have received much interest and effort globally to combat the current pandemic and to prevent such a situation in the future. Currently used vaccines for influenza are administered mainly parenterally and include live attenuated reassortant viruses, conventional inactivated whole viral antigens, or split-virus vaccines. Although some of these vaccines have proven to be quite effective, the manufacturing of these vaccines involves several technical and safety issues (21). Furthermore, the production of currently available influenza vaccines often requires high-level biocontainment facilities, an additional hurdle that limits the advancement of present vaccines.Vaccines containing purified recombinant viral proteins have recently gained special attention due to their ease of production without any safety concerns (25). Recombinant hemagglutinin (rHA) subunit vaccines produced in baculovirus-insect cell expression systems have been extensively tested and evaluated in humans (29, 30). Baculovirus-derived rHA subunit vaccines administered parenterally are safe and immunogenic in animals and humans. Along with its success in recombinant protein vaccines, baculovirus surface display technology allows us to present large complex proteins on the baculovirus envelope in its native antigenic conformation, resulting in good stability and a longer half-life in the host (18, 14, 8).Along with a suitable antigen, the route of administration of the vaccine has a profound effect in controlling mucosally acquired infections such as influenza. Vaccination via the mucosal route stimulates both systemic and mucosal immune responses (16). Oral and intranasal vaccines are the two main options for mucosal administration. Intranasal vaccines would have a detrimental effect on persons with asthma, reactive airway disease, and other chronic pulmonary or cardiovascular disorders (4). Oral vaccines therefore seem to be the safest alternative (13). Moreover, there is evidence to prove the ability of oral vaccination to prevent infection of the lungs (23) and cause transcytosis of the molecule across the cells into the circulation (24).In this report, we describe the construction of recombinant baculovirus under control of the immediate-early promoter 1 (ie1) derived from the white spot syndrome virus (WSSV) genome, which enables the expression of hemagglutinin at the early stage of infection in insect cells, thereby enhancing the display of HA on the baculovirus envelope. Incorporation of more HA into the budding baculovirus particles would improve their efficacy as immunogens. We have studied the efficacy of WSSV ie1-based baculovirus displaying hemagglutinin (BacHA) as an oral vaccine in a mouse model of infection. We have also assessed its efficacy with recombinant cholera toxin B (rCTB) as a mucosal adjuvant. This strategy will enable rapid production of prepandemic vaccines with minimal infrastructure around the world, alleviating the need for high-biosafety facilities, risky inactivation of virulent viruses, and meticulous protein purification procedures.     

病毒感染与宿主细胞DNA损伤应答

病毒感染宿主细胞后,利用细胞内的营养物质和原料进行复制和增殖,同时能引起宿主细胞启动抗病毒免疫应答的防御机制。此外,近年来的研究还表明病毒感染能够引起宿主细胞的DNA损伤应答,该反应是细胞另一种防止病毒入侵的自我保护机制。同时发现,病毒在长期进化过程中形成了不同的机制来对抗宿主细胞的DNA损伤应答,从而消除细胞对其复制和繁殖产生的不利影响。因此,研究和阐述病毒感染后引起宿主细胞DNA损伤应答途径的机制,可使我们采取相应对策选择新的抗病毒靶点,从而有利于新型抗病毒药物的开发。     

Host Genetic Variation Affects Resistance to Infection with a Highly Pathogenic H5N1 Influenza A Virus in Mice

Despite the prevalence of H5N1 influenza viruses in global avian populations, comparatively few cases have been diagnosed in humans. Although viral factors almost certainly play a role in limiting human infection and disease, host genetics most likely contribute substantially. To model host factors in the context of influenza virus infection, we determined the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice and identified genetic elements associated with survival after infection. The lethal dose in these hosts varied by 4 logs and was associated with differences in replication kinetics and increased production of proinflammatory cytokines CCL2 and tumor necrosis factor alpha in susceptible DBA/2J mice. Gene mapping with recombinant inbred BXD strains revealed five loci or Qivr (quantitative trait loci for influenza virus resistance) located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus. In conjunction with gene expression profiling, we identified a number of candidate susceptibility genes. One of the validated genes, the hemolytic complement gene, affected virus titer 7 days after infection. We conclude that H5N1 influenza virus-induced pathology is affected by a complex and multigenic host component.The last 10 years have witnessed a spread of highly pathogenic H5N1 avian influenza A virus from Southeast Asia into Europe and Africa, killing millions of chickens and ducks. Mammalian species including tigers, cats, dogs, and humans have also been infected with H5N1 virus, causing severe and often fatal disease. Excess mortality in humans was associated with high pharyngeal viral loads and increased cytokine and chemokine production (12). Some evidence suggests that genetic variation among infected hosts contributes to H5N1 infection and pathogenesis. Compared to the many millions of chickens and ducks that have been infected by H5N1 virus, relatively few humans have been infected. Were these individuals genetically predisposed, and therefore, did they have a greater risk of getting infected by the currently circulating H5N1 influenza viruses? Also, among the identified clusters of human H5N1 virus infections, more than 90% of the cases have occurred in genetically related family members, suggesting a possible genetic susceptibility to infection or severe disease (33). Recently, genetic relatedness was shown to be a significant risk factor for severe disease resulting from H3N2 influenza virus infection (2). However, other recent studies either have been unable to confirm the effect of genetic variation on the outcome and severity of influenza A virus infection (19) or have challenged the role of host genetics in H5N1 virus clusters (36).Genetic polymorphisms in the infected host affect microbial pathogenesis. In some host-pathogen studies, individual genes strongly regulated disease susceptibility or severity. For example, a 32-amino-acid deletion in the CCR5 product has been associated with increased resistance to human immunodeficiency virus infection (26), and more recently, a single amino acid change in the TLR3 product was associated with herpes simplex virus-induced encephalitis (50). Despite these examples, most host-pathogen interactions are more complex and modified by several genetic determinants. In the mouse model, disease severity after infection with viruses, bacteria, or parasites is frequently caused by multiple genetic differences, each affecting the outcome of the disease (3, 7, 8, 17, 47). Genetic modifiers that are associated with increased susceptibility to influenza virus infection or disease are mostly unknown. In humans, the duration of virus shedding was reduced in HLA-A2⁺ individuals, possibly as a result of a stronger cellular immune response (9). In mice, the resistance to influenza virus infection was mapped to the MX1 protein (39, 44, 46). The human MX1 protein also restricts viral replication, but its efficacy depends on the virus strain (13).Although much work is being done to define the viral factors affecting H5N1 influenza virus pathogenesis, little has been done to elucidate the effect of host genetics on H5N1 disease outcome. This study was initiated to assess the effect of the host''s genetic variation on H5N1 influenza virus pathogenesis and to provide the first clues about which host genes are responsible for the increased pathogenesis of H5N1 virus infection. Genome-wide linkage analysis using BXD recombinant inbred (BXD RI) strains was performed to identify areas on the chromosome that contribute to the difference in susceptibility to H5N1 virus seen between C57BL/6J and DBA/2J mice.     

Immunization against a Saccharide Epitope Accelerates Clearance of Experimental Gonococcal Infection

The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a T_H1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with currently available antibiotics.     

Chronic Trichuris muris Infection in C57BL/6 Mice Causes Significant Changes in Host Microbiota and Metabolome: Effects Reversed by Pathogen Clearance

Trichuris species are a globally important and prevalent group of intestinal helminth parasites, in which Trichuris muris (mouse whipworm) is an ideal model for this disease. This paper describes the first ever highly controlled and comprehensive investigation into the effects of T. muris infection on the faecal microbiota of mice and the effects on the microbiota following successful clearance of the infection. Communities were profiled using DGGE, 454 pyrosequencing, and metabolomics. Changes in microbial composition occurred between 14 and 28 days post infection, resulting in significant changes in α and β- diversity. This impact was dominated by a reduction in the diversity and abundance of Bacteroidetes, specifically Prevotella and Parabacteroides. Metabolomic analysis of stool samples of infected mice at day 41 showed significant differences to uninfected controls with a significant increase in the levels of a number of essential amino acids and a reduction in breakdown of dietary plant derived carbohydrates. The significant reduction in weight gain by infected mice probably reflects these metabolic changes and the incomplete digestion of dietary polysaccharides. Following clearance of infection the intestinal microbiota underwent additional changes gradually transitioning by day 91 towards a microbiota of an uninfected animal. These data indicate that the changes in microbiota as a consequence of infection were transitory requiring the presence of the pathogen for maintenance. Interestingly this was not observed for all of the key immune cell populations associated with chronic T. muris infection. This reflects the highly regulated chronic response and potential lasting immunological consequences of dysbiosis in the microbiota. Thus infection of T. muris causes a significant and substantial impact on intestinal microbiota and digestive function of mice with affects in long term immune regulation.     

Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β₃ (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table ​(Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV

CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.     

Tissue-Protective Effects of NKG2A in Immune-Mediated Clearance of Virus Infection

Virus infection triggers a CD8⁺ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8⁺ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1^b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8⁺ T cells. We have previously shown that Qa1^b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8⁺ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8⁺ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8⁺ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8⁺ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8⁺ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.     

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*^/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*^/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8⁺ T cells in lungs and draining lymph nodes of Nox1*^/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*^/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.     

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.For productive viral protein expression, viruses have to compete for and hijack the host translational machinery (45). Some viruses such as poliovirus, vesicular stomatitis virus (VSV), and influenza virus selectively antagonize the translation apparatus to shut off host translation, resulting in the release of ribosomes from host mRNAs and the inhibition of antiviral responses. On the other hand, the host cell can counteract through antiviral mechanisms to shutdown viral translation. For instance, viral RNA replication intermediates can trigger PKR, leading to an inhibition of overall translation. To bypass the block in translation, viruses have evolved unique mechanisms to preferentially recruit the ribosome for viral protein synthesis. Thus, the control of the translational machinery during infection is a major focal point in the battle between the host and the virus and often, elucidation of these viral translational shutoff strategies reveals key targets of translational regulation.The majority of cellular mRNAs initiate translation through the recruitment of the cap-binding complex, eukaryotic translation initiation factor 4F (eIF4F), to the 5′ cap of the mRNA (56). eIF4F consists of the cap-binding protein eIF4E, the RNA helicase, eIF4A, and the adaptor protein eIF4G. eIF4G acts as a bridge to join eIF4E and the 40S subunit via eIF3. With the ternary eIF2-Met-tRNA_i-GTP complex bound, the 40S subunit scans in a 5′-to-3′ direction until an AUG start codon is encountered. Here, eIF5 mediates GTP hydrolysis on the ternary complex, releasing the eIFs and subsequently leading to 60S subunit joining to assemble an elongation-competent 80S ribosome. The ternary eIF2-Met-tRNA_i-GTP complex is reactivated for another round of translation by exchange of GDP for GTP, which is mediated by the guanine nucleotide exchange factor, eIF2B. The 3′ poly(A) tail of the mRNA also stimulates translational initiation by binding to the poly(A) binding protein (PABP), which in turn interacts with eIF4G at the 5′end, resulting in a circularized mRNA. PABP has been proposed to enhance eIF4E affinity for the 5′cap and promote 60S joining, indicating that PABP functions at multiple steps of translational initiation (33).A common tactic viruses use to inhibit host translation is to selectively target eIFs. One of the best studied is the cleavage of eIF4G by viral proteases during picornavirus infection. In humans, two isoforms, eIF4GI and eIF4GII, are cleaved early in poliovirus infection by the viral protease 2A, where cleavage of eIFGII correlates more precisely with host translation shutoff (20). Cleavage of eIF4G produces an amino-terminal fragment that binds to eIF4E and a C-terminal fragment that binds to eIF4A and eIF3 (26, 39, 42). PABP is also cleaved by the viral protease 3C during poliovirus infection, thus contributing to shutoff of both host and viral translation and thereby enabling the switch from viral translation to replication (3, 31, 38). Another major target is the availability of the cap-binding protein eIF4E, which is regulated by binding to the repressor protein 4E-BP (21, 41). 4E-BP and eIF4G compete for an overlapping site on eIF4E (42). In its hypophosphorylated state, 4E-BP binds to and sequesters eIF4E, preventing eIF4G recruitment. Dephosphorylation and activation of 4E-BP has been observed during poliovirus, encephalomyocarditis (EMCV), and VSV infections (7, 18).During virus infection, host antiviral responses are triggered that also inhibit translation to counteract viral protein synthesis. An integral antiviral response is phosphorylation at Ser⁵¹ of eIF2α, which reduces the pool of the ternary complex by blocking the eIF2B-dependent exchange of GDP to GTP. In mammals, four known eIF2α kinases exist including the endoplasmic reticulum (ER)-stress-inducible PERK, GCN2, which senses the accumulation of deacylated tRNAs during amino acid starvation conditions; the heme-regulated kinase HRI; and the interferon-inducible double-stranded RNA-binding PKR (64). In mammalian cells, PKR is activated by binding to double-stranded viral RNA replication intermediates, leading to eIF2α phosphorylation and inhibition of overall host and viral translation. PERK and GCN2 have also been shown to be activated during virus infections by VSV and members of the alphavirus family (2, 6, 43, 65, 79). Often, viruses rely on the ER for synthesis and proper folding of viral proteins. The large burden on the ER activates PERK to phosphorylate eIF2α, thereby inhibiting global protein synthesis to reduce the load on the ER (23). Some viruses such as HCV and herpes simplex viruses have adapted to responses that induce eIF2α phosphorylation by producing viral proteins that counteract PKR or modulate the ER stress response (27, 76). Thus, virus infection can trigger several eIF2α kinases that lead to translational shutoff to counteract viral protein synthesis.To circumvent these translation blocks, viruses such as poliovirus and hepatitis C virus utilize internal ribosome entry sites (IRES), which are RNA elements that directly recruit ribosomes in a cap-independent manner and require only a subset of canonical eIFs (15, 25). It is generally thought that IRES-containing viral mRNAs can be translated under conditions when specific eIFs are compromised during infection. Except for a few cases, the specific mechanisms and factors that lead to IRES stimulation is poorly understood. For example, poliovirus and the related EMCV possess an IRES that allows viral translation despite cleavage of eIF4G during infection or inhibiting eIF4E by 4E-BP binding. This type of IRES can still bind to the central domain of eIF4G and mediate 40S subunit recruitment (11, 37, 57).One of the most unique and simplest IRES is found within the intergenic region (IGR) of the Dicistroviridae family (for extensive reviews, see references 28, 36, and 49). Members of this family include the cricket paralysis virus (CrPV), drosophila C virus (DCV), taura syndrome virus, the Plautia stali intestine virus (PSIV), the Rhopalosiphum padi virus (RhPV), and several bee viruses such as the black queen cell virus and the Israeli acute paralysis virus, which has been recently linked to colony collapse disorder (10). The dicistroviruses encode a positive-strand 8- to 10-kb single-stranded RNA genome, which contains two main open reading frames, ORF1 and ORF2, encoding the nonstructural and structural proteins, respectively, separated by an IGR (see Fig. ​Fig.1A).1A). The 5′ end of the CrPV RNA is linked to the viral protein VpG and the 3′ end contains a poly(A) tail (16). Radiolabeling of intracellular RNA in infected cells reveals no subgenomic RNA species smaller than the full-length genomic RNA, and this has been supported by Northern blot analysis (16, 81). Translation of ORF2 is directed by the IGR IRES, whereas ORF1 expression is mediated by an IRES within the 5′ untranslated region (5′UTR) (35, 67, 81, 82). Remarkably, the IGR IRES element can directly recruit the ribosome independently of eIFs or the initiator Met-tRNA_i (29, 30, 54, 80). Furthermore, the IRES occupies the P-site of the ribosome to initiate translation from the ribosomal A-site encoding non-AUG codon (35, 81). Extensive biochemical and structural analyses from several groups have revealed that the IGR IRES mimics a tRNA that occupies the mRNA cleft of the ribosome and sets the ribosome into an elongation state (9, 29, 30, 34, 51, 55, 58, 68, 72, 83). Using reporter constructs, it has also been demonstrated that CrPV IGR IRES-mediated translation is active under a number of cellular conditions when the activity of the ternary complex eIF2-Met-tRNA_i-GTP is compromised (17, 63, 78, 80). Because IGR IRES-mediated translation does not require initiation factors, the IRES can direct translation under a number of cellular conditions when the activity of multiple eIFs is compromised (12). Although the majority of studies have focused on the IGR IRES of CrPV, PSIV, and TSV, it is predicted that the IGRs within this viral family all function similarly based on the predicted conserved RNA structures (28, 36, 49). In contrast, only the 5′UTR IRES mechanism of RhPV has been studied in detail (77). Despite the wealth of studies on the mechanics of these IRES, the mechanisms that lead to translational shutoff during dicistrovirus infection and the interaction of dicistrovirus with the host machinery to allow virus production have been relatively unexplored.Open in a separate windowFIG. 1.Kinetics of host protein synthesis and viral protein expression in CrPV-infected Drosophila S2 cells. (A) Genomic arrangement of the CrPV RNA. The viral open reading frames, ORF1 and ORF2, that encode nonstructural (NS) and structural (S) proteins, respectively, are shown, which are separated by the intergenic internal ribosome entry site (IGR IRES). Translation of ORF1 and ORF2 is directed by the 5′UTR IRES and the IGR IRES, respectively. The first amino acid of ORF2 directed by the IGR IRES is encoded by a GCU alanine codon. (B) Autoradiography of protein lysates resolved on a SDS-12% PAGE gel. The protein lysates were collected from S2 cells that were untreated (U), mock infected (M), CrPV infected (5 FFU/cell), or thapsigargin treated (Tg; 0.4 μM) for the indicated times (h p.i.) and metabolically labeled with [³⁵S]methionine for 30 min at each time point. The migration of proteins with known molecular masses is shown on the left. The expression of detectable nonstructural (NS) and structural (S) proteins is denoted. (C) Quantitation of host protein synthesis during CrPV infection. To calculate the host translation at each time point, the amount of radioactivity of the bands between 55 and 70 kDa in panel A was quantitated by using ImageQuant, and the percent translation was calculated at each time point of virus infection or thapsigargin treatment compared to the mock infection. Shown are averages (± the standard deviation) from at least three independent experiments. (D) Immunoblots of viral ORF1 and ORF2 during CrPV infection at various times postinfection (h p.i.). Antibodies were raised against peptides within ORF1 and ORF2. The expression of ORF1 and ORF2 was quantitated by a LI-COR Odyssey system, plotted against time of infection, and normalized to the amount of ORF1 or ORF2 expression at 6 h p.i. (100%). As a comparison, viral RNA synthesis as detected by Northern blot analysis (see Fig. ​Fig.2B)2B) is plotted on the same graph.Previous studies have shown that the CrPV and the related DCV can infect a wide range of insect hosts, including the Drosophila melanogaster S2 cell line (60, 69). In the present study, we have explored how CrPV infection leads to host translational shutoff in S2 cells. Two steps of translational initiation are targeted during CrPV infection. First, the interaction of deIF4G with deIF4E is disrupted early in infection and remains dissociated during the course of infection. Second, deIF2α is phosphorylated at a time that lags after the initial host translational shutoff during infection. Premature phosphorylation of deIF2α early in infection inhibited translation directed by the 5′UTR IRES, but IGR IRES-mediated translation remained relatively resistant. These results support the model that multiple mechanisms, including impairment of deIF4F complex formation and induction of deIF2α phosphorylation, contribute to the host translational shutoff during CrPV infection. The inhibition of host translation and the release of ribosomes from host mRNAs ensures that translation mediated by the 5′UTR and IGR IRES is optimal to produce sufficient viral nonstructural and structural proteins for proper CrPV maturation and assembly.