Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE: Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

Baculovirus cyclobutane pyrimidine dimer photolyases show a close relationship with lepidopteran host homologues.

Cyclobutane pyrimidine dimer (CPD) photolyases repair ultraviolet (UV)-induced DNA damage using blue light. To get insight in the origin of baculovirus CPD photolyase (phr) genes, homologues in the lepidopteran insects Chrysodeixis chalcites, Spodoptera exigua and Trichoplusia ni were identified and characterized. Lepidopteran and baculovirus phr genes each form a monophyletic group, and together form a well-supported clade within the insect photolyases. This suggests that baculoviruses obtained their phr genes from an ancestral lepidopteran insect host. A likely evolutionary scenario is that a granulovirus, Spodoptera litura GV or a direct ancestor, obtained a phr gene. Subsequently, it was horizontally transferred from this granulovirus to several group II nucleopolyhedroviruses (NPVs), including those that infect noctuids of the Plusiinae subfamily.

Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1.

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146 bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

Translation of both 5'TOP and non-TOP host mRNAs continues into the late phase of Baculovirus infection.

Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2alpha from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a 5' terminal oligopyrimidine (TOP) tract classified the lepidopteran rpL15 transcript as a TOP mRNA. For eEF2, two types of transcripts were observed, one of which had a 5'TOP tract. The transcript levels for rpL15, eEF2 and eIF2alpha decreased following baculovirus infection. Polysome analysis showed that the corresponding mRNAs remained to be translated until at least 16 h post-infection for both TOP and non-TOP mRNAs. Baculovirus-induced host shut-off therefore appears to be regulated at the level of RNA abundance rather than at the translational level.

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

Effect of baculovirus infection on the mRNA and protein levels of the Spodoptera frugiperda eukaryotic initiation factor 4E.

The cDNA sequence of eukaryotic translation initiation factor eIF4E was derived from a Spodoptera frugiperda cDNA library. Eight tryptophan residues, typical for eIF4E, are strictly conserved in the encoded 210 amino acid protein. A polyclonal antiserum detected a 26 kDa protein in lepidopteran cell lines, but not in dipteran cells. Sf21 cells have a single eIF4E gene copy, which is transcribed into a 1500 nt transcript. Infection with AcMNPV resulted in a decrease in eIF4E mRNA starting between 12 and 24 h postinfection (p.i.), while reduced eIF4E protein levels were observed at 48 h p.i. Two forms of eIF4E were recognized that differed in their iso-electric point, of which the relative abundance did not change during infection. Mutagenesis experiments using recombinant baculoviruses revealed that the variation in mobility between these two forms did not result from a difference in the phosphorylation state of Ser-202, the serine residue that corresponds with the eIF4E phosphorylation site in mammalian eIF4E.

Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells.

The 3' untranslated region (UTR) is an important element that determines the level of recombinant protein expression via baculovirus vectors. Previous work using chloramphenicol acetyl transferase as reporter has shown that p10-promoter based baculovirus vectors with the authentic p10 3' UTR resulted in higher expression levels than vectors carrying an SV40 early terminator, as part of a lacZ selection cassette. To examine whether a similar increase in expression levels could be obtained for baculovirus-expressed glycoproteins, the classical swine fever virus E2 antigen was used as a model. With the authentic p10 3' UTR higher levels of E2 transcript were found than in the presence of the SV40 terminator. This higher number of transcripts was accompanied by elevated levels of intracellular, non-glycosylated E2 protein. However, the levels of intracellular glycosylated forms of E2 and of extracellular E2 were similar for both type of terminators. These results show that translation of the recombinant mRNA is not the rate limiting step in the expression of glycoproteins, but the downstream processing and secretion of the translation products.

Specificity of polyhedrin in the generation of baculovirus occlusion bodies.

The role of polyhedrin in the occlusion of virions was studied by substituting two heterologous polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.

Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species.

Eukaryotic initiation factor eIF5A is essential for cell viability and contains a characteristic post-translational modification of a specific lysine residue into a hypusine. cDNAs with similarity to eIF5A sequences were derived from Spodoptera exigua and S. frugiperda cDNA libraries. The deduced amino acid sequences are identical for both species and predict a protein with a molecular mass of 17.5 kDa. The Drosophila melanogaster eIF5A cDNA sequence was retrieved from the Drosophila EST Project. The predicted protein is 80% similar to Spodoptera eIF5A. A single eIF5A gene copy is present in the S. frugiperda genome, which is transcribed into four different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decline of all four transcripts already at 12 h after infection. In contrast, the eIF5A protein was fairly stable up to 48 h post infection.

The single-nucleocapsid nucleopolyhedrovirus of Buzura suppressaria encodes a P10 protein.

The p10 gene of Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was identified by virtue of its localization downstream from the Autographa californica (Ac) MNPV p26 homologue. The BusuNPV p10 gene encodes a protein of 94 amino acids. The amino acid sequence contains domains characteristic of baculovirus P10 proteins, e.g. a coiled-coil domain, a proline-rich motif and a positively charged C terminus. The highest amino acid homologies were found with the Spodoptera littoralis (Spli) NPV and Spodoptera exigua (Se) MNPV P10 proteins. An AcMNPV recombinant expressing the BusuNPV P10 formed fibrillar structures in the cytoplasm of Spodoptera frugiperda cells. BusuNPV P10 could not fully replace AcMNPV P10 in its nuclear disintegration function, since polyhedra were not efficiently liberated from infected cells late in infection. The BusuNPV p26 gene encodes a protein of 263 amino acid residues with 70% amino acid similarity with SeMNPV P26. Downstream of the BusuNPV p10 gene, the gene for the occlusion-derived virus protein ODVP-6e is located. This is unlike the situation in many other NPVs, including SeMNPV, where the p10 gene neighbours the p74 gene. The data presented here suggest that although the p10 gene is not conserved in sequence, evolutionary pressure preserves the structure of P10 and hence its function. These data also indicate that all NPVs, MNPVs as well as SNPVs, contain this gene.

Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae.

A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.

Specificity of baculovirus p10 functions.

Three functional domains in baculovirus p10 proteins have been postulated for aggregation, nuclear disintegration, and fibrillar structure formation (Van Oers et al., J. Gen. Virol. 74, 563-574, 1993). To study the specificity of these functions, a recombinant Autographa californica nuclear polyhedrosis virus (AcCR1) was constructed in which the coding sequence of the p10 gene was replaced with the p10 sequence of the distantly related Spodoptera exigua (Se) MNPV. In AcCR1-infected cells the SeMNPV p10 protein was produced at similarly high levels as AcMNPV p10 in wild type (wt) AcMNPV infections. Formation of fibrillar structures occurred in a similar fashion in SeMNPV and AcCR1-infected cells. Hence, the SeMNPV p10 protein retained the ability to associate into fibrillar structures when expressed in an otherwise AcMNPV context. Mixed infection with wt AcMNPV and AcCR1 indicated that only p10 proteins of the same species aggregate and that these aggregates associate into fibrillar structures. In contrast to S. exigua cells infected with AcMNPV or SeMNPV, S. exigua cells infected with AcCR1 failed to release polyhedra. This result indicated that interaction of p10 with at least one virus-specific factor is required for nuclear disintegration.

Nucleotide sequence and transcriptional analysis of the p10 gene of Spodoptera exigua nuclear polyhedrosis virus.

The p10 gene of Spodoptera exigua multiple nuclear polyhedrosis virus (SeMNPV) was localized on the XbaI fragment H (5.1 kb) of the physical map of the viral genome. The coding sequence of the SeMNPV p10 gene is 264 nucleotides (nt) long corresponding to a predicted protein of 88 amino acids with an MHF of 9607. The SeMNPV p10 protein showed only limited amino acid identity (39% and 26%, respectively) to those of Orgyia pseudotsugata MNPV (OpMNPV) and Autographa californica MNPV (AcMNPV) and thus appears less conserved than other viral proteins. The SeMNPV p10 gene was expressed by a transcript of approximately 450 nt, which started in the conserved baculovirus late gene promoter motif TAAG. The leader of the SeMNPV p10 transcript was AT-rich (92%) and at 36 nt was the shortest leader of all baculovirus major late genes reported so far. The SeMPNV p10 transcript terminated 6 nt downstream from a putative poly(A) signal sequence (AATAAA); the latter was 61 nt downstream of the translational stop codon TAA. Upstream and downstream of the p10 gene, partial putative ORFs were found that showed significant amino acid sequence identity to the baculovirus p26 and p74 proteins. It is concluded that the region of SeMNPV DNA containing the p10 gene is collinear with the corresponding regions in the AcMNPV and OpMNPV genomes.

Functional domains of the p10 protein of Autographa californica nuclear polyhedrosis virus.

Distinct functional domains in the Autographa californica nuclear polyhedrosis virus p10 protein were identified by analysis of p10 mutants. When up to 15 amino acids from the carboxy terminus were deleted, truncated p10 proteins were found in both the nucleus and the cytoplasm of infected cells, but formed no fibrillar structures. This suggested that the positively charged carboxy terminus is not required for nuclear or cytoplasmic localization of p10 protein, but is involved in protein-protein interactions leading to assembly of the p10 protein into fibrillar structures. Absence of the p10 protein prevented the release of polyhedra from infected cells, caused by impaired nuclear disintegration. This function of the p10 protein appears to be located between amino acid residues 52 and 79. The amino-terminal half of the p10 protein has already been implicated in the self-aggregation of this protein. Thus fibrillar structure formation, nuclear disintegration and intermolecular p10 protein interactions seem to be three separate functions of the p10 protein and these functions are located in distinct domains of the protein. The mutants expressing truncated p10 proteins were impaired in electron-dense spacer formation but polyhedron envelopes were still formed. This result suggested that the formation of electron-dense spacers is not a prerequisite for the formation of polyhedron envelopes.

Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of polyhedrin gene expression.

Two major late proteins, polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the polyhedrin gene, several recombinants were constructed with various deletions in the polyhedrin gene. The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and polyhedrin-driven expression. None of the deletions in the polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. This suggested that the transcriptional and/or translational activity of the p10 and polyhedrin gene are independently regulated. To compare the level of polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.