Revertant Viruses Research Articles

Regulation viral RNA transcription and replication by higher-order RNA structures within the nsp1 coding region of MERS coronavirus

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.

Cytomegalovirus inhibitors of programmed cell death restrict antigen cross-presentation in the priming of antiviral CD8 T cells.

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.

Identification and quantitation of multiple variants in RNA virus genomes.

The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances >5 kb. As proof of concept, high-quality viral RNA of the Dengue 2 component of Takeda's tetravalent dengue vaccine candidate (TDV-2) was used to develop a reverse transcription-polymerase chain reaction protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long-read sequencing was validated with multiple clone-derived TDV-2 revertant variants and four complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in the differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single-genome molecule, which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.

Identification of a critical role for ZIKV capsid α3 in virus assembly and its genetic interaction with M protein.

Flaviviruses such as Zika and dengue viruses are persistent health concerns in endemic regions worldwide. Efforts to combat the spread of flaviviruses have been challenging, as no antivirals or optimal vaccines are available. Prevention and treatment of flavivirus-induced diseases require a comprehensive understanding of their life cycle. However, several aspects of flavivirus biogenesis, including genome packaging and virion assembly, are not well characterized. In this study, we focused on flavivirus capsid protein (C) using Zika virus (ZIKV) as a model to investigate the role of the externally oriented α3 helix (C α3) without a known or predicted function. Alanine scanning mutagenesis of surface-exposed amino acids on C α3 revealed a critical CN67 residue essential for ZIKV virion production. The CN67A mutation did not affect dimerization or RNA binding of purified C protein in vitro. The virus assembly is severely affected in cells transfected with an infectious cDNA clone of ZIKV with CN67A mutation, resulting in a highly attenuated phenotype. We isolated a revertant virus with a partially restored phenotype by continuous passage of the CN67A mutant virus in Vero E6 cells. Sequence analysis of the revertant revealed a second site mutation in the viral membrane (M) protein MF37L, indicating a genetic interaction between the C and M proteins of ZIKV. Introducing the MF37L mutation on the mutant ZIKV CN67A generated a double-mutant virus phenotypically consistent with the isolated genetic revertant. Similar results were obtained with analogous mutations on C and M proteins of dengue virus, suggesting the critical nature of C α3 and possible C and M residues contributing to virus assembly in other Aedes-transmitted flaviviruses. This study provides the first experimental evidence of a genetic interaction between the C protein and the viral envelope protein M, providing a mechanistic understanding of the molecular interactions involved in the assembly and budding of Aedes-transmitted flaviviruses.

Virally encoded interleukin-6 facilitates KSHV replication in monocytes and induction of dysfunctional macrophages.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi's sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Using scRNA-seq, we demonstrate that KSHV preferentially infects CD14+ monocytes, sustains viral lytic replication through the viral interleukin-6 (vIL-6), which activates STAT1 and 3, and induces an inflammatory gene expression program. To study the role of vIL-6 in monocytes upon KSHV infection, we generated recombinant KSHV with premature stop codon (vIL-6(-)) and its revertant viruses (vIL-6(+)). Infection of the recombinant viruses shows that both vIL-6(+) and vIL-6(-) KSHV infection induced indistinguishable host anti-viral response with STAT1 and 3 activations in monocytes; however, vIL-6(+), but not vIL-6(-), KSHV infection promoted the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL-6(+) KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL-6(-) KSHV infection or uninfected control. Notably, a viral nuclear long noncoding RNA (PAN RNA), which is required for sustaining KSHV gene expression, was substantially reduced in infected primary monocytes upon vIL-6(-) KSHV infection. These results highlight the critical role of vIL-6 in sustaining KSHV transcription in primary monocytes. Our findings also imply a clever strategy in which KSHV utilizes vIL-6 to secure its viral pool by expanding infected monocytes via differentiating into longer-lived dysfunctional macrophages. This mechanism may facilitate KSHV to escape from host immune surveillance and to support a lifelong infection.

From vaccine to pathogen: Modeling Sabin 2 vaccine virus reversion andevolutionary epidemiology inMatlab, Bangladesh.

The oral poliovirus vaccines (OPVs) are one of the most effective disease eradication tools in public health. However, the OPV strains are genetically unstable and can cause outbreaks of circulating, vaccine-derived Type 2 poliovirus (cVDPV2) that are clinically indistinguishable from wild poliovirus (WPV) outbreaks. Here, we developed a Sabin 2 reversion model that simulates the reversion of Sabin 2 to reacquire a WPV-like phenotype based on the clinical differences in shedding duration and infectiousness between individuals vaccinated with Sabin 2 and those infected with WPV. Genetic reversion is informed by a canonical reversion pathway defined by three gatekeeper mutations (A481G, U2909C, and U398C) and the accumulation of deleterious nonsynonymous mutations. Our model captures essential aspects of both phenotypic and molecular evolution and simulates transmission using a multiscale transmission model that consolidates the relationships among immunity, susceptibility, and transmission risk. Despite rapid Sabin 2 attenuation reversal, we show that the emergence of a revertant virus does not guarantee a cVDPV2 outbreak. When simulating outbreaks in Matlab, Bangladesh, we found that cVDPV2 outbreaks are most likely in areas with low population-level immunity and poor sanitation. In Matlab, our model predicted that declining immunity against Type 2 poliovirus following the cessation of routine OPV vaccination was not enough to promote cVDPV2 emergence. However, cVDPV2 emergencedepended on the average viral exposure dose per contact, which was modeled as a combination of the viral concentration per fecal gram and the average fecal-oral dose per contact. These results suggest that cVDPV2 emergence risk can be mitigated by reducing the amount of infectious fecal material individuals are exposed to. Thus, a combined strategy of assessing and improving sanitation levels in conjunction with high-coverage vaccination campaigns could limit the future cVDPV2 emergence.

Equid herpesvirus type 3 infection produces membrane-associated and secreted forms of glycoprotein G that are not required for efficient cell-to-cell spread of the virus in vitro.

The ORF 70 gene of equid alphaherpesvirus type 3 (EHV-3) encodes glycoprotein G (gG), which is conserved in the majority of alphaherpesviruses. This glycoprotein is located in the viral envelope and has the characteristic of being secreted into the culture medium after proteolytic processing. It modulates the antiviral immune response of the host by interacting with chemokines. The aim of this study was to identify and characterize EHV-3 gG. By constructing viruses with HA-tagged gG, it was possible to detect gG in lysates of infected cells, their supernatants, and purified virions. A 100-, 60-, and 17-kDa form of the protein were detected in viral particles, while a 60-kDa form was identified in supernatants of infected cells. The role of EHV-3 gG in the viral infection cycle was assessed by the construction of a gG-minus EHV-3 mutant and its gG-positive revertant. When growth characteristics in an equine dermal fibroblast cell line were compared, the plaque size and the growth kinetics of the gG-minus mutant were similar to those of the revertant virus, suggesting that EHV-3 gG does not play a role in direct cell-to-cell transmission or virus proliferation of EHV-3 in tissue culture. The identification and characterization of EHV-3 gG described here provide a solid background for further studies to assess whether this glycoprotein has a function in modulating the host immune response.

Deleting UL49.5 in duck plague virus causes attachment, entry and spread defects

Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL49.5 protein (pUL49.5) is homologue of glycoprotein N (gN), which is conserved in herpesviruses. UL49.5 homologues are known to be involved in processes such as immune escape, virus assembly, viral fusion, transporter associated with antigen processing (TAP) inhibition and degradation, and maturation and incorporation of glycoprotein M. However, few studies have focused on the role of gN in the early stage of virus infection cells. In this study, we determined that DPV pUL49.5 was distributed in the cytoplasm and colocalized with the endoplasmic reticulum (ER). Moreover, we found that DPV pUL49.5 was a virion component and nonglycosylated protein. To better explore its function, BAC-DPV-ΔUL49.5 was constructed, and its attachment was only approximately 25 % of the revertant virus. Additionally, the penetration ability of BAC-DPV-ΔUL49.5 has only reached 73 % of the revertant virus. The plaque sizes produced by the UL49.5-deleted virus were approximately 58 % smaller than those produced by the revertant virus. Deleting UL49.5 mainly resulted in attachment and cell-to-cell-spread defects. Taken together, these findings suggest important roles for DPV pUL49.5 in viral attachment, penetration and spread.

Research Note: Duck plague virus pUL48 is a late protein that plays an important role in viral replication

Duck plague virus (DPV) pUL48 is a homologous of herpes simplex virus VP16, and some studies have shown that VP16 is essential for viral replication and proliferation, but there are few studies on DPV pUL48. Therefore, in order to study the function of pUL48 protein, we constructed a UL48-deleted mutant (DPV-BAC-∆UL48) that completely reemoved the UL48 gene from the DPV BAC genome and the revertant virus (DPV-BAC-∆UL48R) by using the 2-step red recombination system. Compared with the parental virus (DPV-BAC) and the revertant virus, the titer of UL48-deleted mutant was reduced by more than 38.2%, and the efficiency of producing infectious virions was significantly reduced. In addition, the average size of plaques produced by UL48-deleted mutant was about 30% smaller than that of the parental and revertant viruses, suggesting that pUL48 protein affected the cell-to-cell transmission of DPV. Finally, pharmacological inhibition assay showed that pUL48 is a late protein of DPV. In this study, we found that UL48, as a late gene, plays an important role in viral replication by affecting the formation of DPV infectious virion, virus cell-to-cell transmission, and viral genome transcription, which may provide some help for the study of the function of DPV pUL48 protein and the prevention and control of DPV.

Bovine Herpesvirus-1 Glycoprotein M Mediates the Translocation to the Golgi Apparatus and Packaging of VP8.

VP8, the most abundant tegument protein of bovine herpesvirus-1 (BoHV-1), plays an important role in viral replication. According to our previous studies, VP8 localizes to the Golgi apparatus of BoHV-1-infected cells where it can be packaged into the virus; however, Golgi localization of VP8 does not occur outside of the context of infection. The goal of this study was to identify the viral factor(s) involved in the tropism of VP8 towards the Golgi. VP8 was found to interact with glycoprotein M (gM), and the VP8 and gM domains that are essential for this interaction were identified. VP8 and gM colocalized to the Golgi apparatus in BoHV-1-infected cells. In cells co-transfected with VP8- and gM-encoding plasmids, VP8 was also found to be localized to the Golgi, suggesting gM to be sufficient. The localization of VP8 to the Golgi was lost in cells infected with a gM deletion mutant, and the amount of VP8 incorporated into mature virus was significantly reduced. However, with the restoration of gM in a revertant virus, the localization to the Golgi and the amount of VP8 incorporated in the virions were restored. These results indicate that gM plays a critical role in VP8 subcellular localization to the Golgi and packaging into mature virions.

Evaluation of Immune Responses and Protective Efficacy of a Novel Live Attenuated Salmonella Enteritidis Vaccine Candidate in Chickens.

An ideal vaccine for controlling Salmonella infection in chicken flocks should be safe, inducing both humoral and cellular immunity. Live attenuated vaccines against Salmonella Enteritidis (S. Enteritidis) have been used as a potential control method of Salmonella infection in the poultry industry. However, live attenuated vaccines can persistently infect poultry for long periods and can become virulent revertant strains. In this study, we assessed the immune responses and protective efficacy of a temperature-sensitive attenuated S. Enteritidis mutant as a potential vaccine candidate. In addition, we evaluated the combined vaccine administration methods to maximize both humoral and cellular immune responses in chickens induced by the vaccine candidate. Immune responses and protective efficacy were compared between the Oral/IM group, vaccinated using one oral dose at four weeks old and a booster intramuscular dose at seven weeks old, and the IM/Oral group, vaccinated using one intramuscular dose at four weeks old and a booster oral dose at seven weeks old. The Oral/IM group showed stronger immune responses than those of the IM/Oral group. Spleens from the Oral/IM group showed a promising tendency of reduction of challenged Salmonella compared with those of other groups. Overall, the results indicated that the S. Enteritidis mutant strain is a promising live attenuated vaccine candidate with good efficacy.

The protein encoded by the duck plague virus UL14 gene regulates virion morphogenesis and affects viral replication

To investigate the pivotal roles of the duck plague virus (DPV) tegument protein UL14 in viral replication, we generated 2 mutated viruses of DPV by using the bacterial artifcial chromosome system, the UL14-null mutant virus (CHv-BAC-ΔUL14) and the corresponding revertant virus (CHv-BAC-ΔUL14R). We found that the CHv-BAC-ΔUL14 viruses exhibited impaired virion morphogenesis in transmission electron microscopy (TEM) studies. Furthermore, CHv-BAC-ΔUL14 exhibited a plaque size reduction in duck embryo fibroblasts (DEFs). Finally, CHv-BAC-ΔUL14 exhibited a significant viral growth defect. Taken together, our findings suggest that DPV UL14 protein regulates viral morphogenesis for efficient viral replication.

Virus-encoded microRNA-M7 restricts early cytolytic replication and pathogenesis of Marek’s disease virus

MicroRNAs (miRNAs) are a class of ∼22 nucleotides non-coding RNAs that are encoded by a wide range of hosts. Viruses, especially herpesviruses, encode a variety of miRNAs that involved in disease progression. Recently, a cluster of virus-encoded miRNAs, miR-M8-M10, have been shown to restrict early cytolytic replication and pathogenesis of Marek’s disease virus (MDV), an oncogenic avian alphaherpesvirus that causes lymphoproliferative disease in chickens. In this study, we specifically dissected the role of miR-M7, a member of cluster miR-M8-M10, in regulating MDV replication and pathogenesis. We found that deletion of miR-M7-5p did not affect the virus plaque size and growth in cell culture. However, compared to parental virus, infection of miR-M7-5p deletion virus significantly increased MDV genome copy number at 5 days post infection, suggesting that miR-M7 plays a role to restrict MDV replication during early cytolytic phase. In addition, our results showed that infection of miR-M7-5p deletion virus significantly enhanced the mortality of chickens, even it induced lymphoid organ atrophy similar to parental and revertant viruses. Taken together, our study revealed that the miR-M7 acts as a repressive factor of MDV replication and pathogenesis.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

Marek's Disease Virus Cluster 3 miRNAs Restrict Virus' Early Cytolytic Replication and Pathogenesis.

Herpesvirus-encoded microRNAs (miRNAs) have been discovered in infected cells; however, lack of a suitable animal model has hampered functional analyses of viral miRNAs in vivo. Marek’s disease virus (MDV) (Gallid alphaherpesvirus 2, GaHV-2) genome contains 14 miRNA precursors, which encode 26 mature miRNAs, grouped into three clusters. In this study, the role of MDV-encoded cluster 3 miRNAs, also known as mdv1-miR-M8-M10, in pathogenesis was evaluated in chickens, the natural host of MDV. Our results show that deletion of cluster 3 miRNAs did not affect virus replication and plaque size in cell culture, but increased early cytolytic replication of MDV in chickens. We also observed that deletion of cluster 3 miRNAs resulted in significantly higher virus reactivation from peripheral blood lymphocytes. In addition, pathogenesis studies showed that deletion of cluster 3 miRNAs resulted in more severe atrophy of lymphoid organs and reduced mean death time, but did not affect the incidence of MDV-associated visceral tumors. We confirmed these results by generating a cluster 3 miRNA revertant virus in which the parental MDV phenotype was restored. To the best of our knowledge, our study provides the first evidence that MDV cluster 3 miRNAs play an important role in modulating MDV pathogenesis.

Deletion of LORF9 but not LORF10 attenuates Marek’s disease virus pathogenesis

Marek’s disease virus (MDV) genome contains a number of uncharacterized long open reading frames (LORF) and their role in viral pathogenesis has not been fully investigated. Among them, LORF9 (MDV069) and LORF10 (MDV071) are locate at the right terminus of the MDV genome unique long region (UL). To investigate their role in MDV pathogenesis, we generated LORF9 or LORF10 deletion and revertant viruses. In vitro growth kinetics results show that both LORF9 and LORF10 are not essential for virus growth in cell culture. However, LORF9, but not LORF10, is involved in MDV early cytolytic replication in vivo, as evidenced by limited viral antigen expression in lymphoid organs of LORF9 deletion virus inoculated chickens. MDV genome copy number data further confirmed that LORF9 is important for MDV replication in spleen during early cytolytic phase. Deletion of LORF9 also partially impairs the replication of MDV in feather follicle epithelium (FFE); however, it can still establish latency and transformation. In addition, pathogenesis studies show that deletion of LORF9, but not LORF10, result in significant reduction of MDV induced mortality and slightly reduce MDV associated tumors of inoculated chickens. Importantly, we confirmed these results with the generation of LORF9 and LORF10 revertant viruses that fully restore the phenotypes of parental MDV. In conclusion, our results show that deletion of LORF9, but not LORF10, significantly impair viral replication in lymphoid organs during early cytolytic phase and attenuate Marek’s disease virus pathogenesis.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

Different Degrees of 5'-to-3' DAR Interactions Modulate Zika Virus Genome Cyclization and Host-Specific Replication.

Mosquito-borne flaviviruses, which include many important human pathogens, such as West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), have caused numerous emerging epidemics in recent years. Details of the viral genome functions necessary for effective viral replication in mosquito and vertebrate hosts remain obscure. Here, using ZIKV as a model, we found that the conserved "downstream of AUG region" (DAR), which is known to be an essential element for genome cyclization, is involved in viral replication in a host-specific manner. Mutational analysis of the DAR element showed that a single-nucleotide mismatch between the 5' DAR and the 3' DAR had little effect on ZIKV replication in mammalian cells but dramatically impaired viral propagation in mosquito cells. The revertant viruses passaged in mosquito cells generated compensatory mutations restoring the base pairing of the DAR, further confirming the importance of the complementarity of the DAR in mosquito cells. We demonstrate that a single-nucleotide mutation in the DAR is sufficient to destroy long-range RNA interaction of the ZIKV genome and affects de novo RNA synthesis at 28°C instead of 37°C, resulting in the different replication efficiencies of the mutant viruses in mosquito and mammalian cells. Our results reveal a novel function of the circular form of the flavivirus genome in host-specific viral replication, providing new ideas to further explore the functions of the viral genome during host adaptation.IMPORTANCE Flaviviruses naturally cycle between the mosquito vector and vertebrate hosts. The disparate hosts provide selective pressures that drive virus genome evolution to maintain efficient replication during host alteration. Host adaptation may occur at different stages of the viral life cycle, since host-specific viral protein processing and virion conformations have been reported in the individual hosts. However, the viral determinants and the underlying mechanisms associated with host-specific functions remain obscure. In this study, using Zika virus, we found that the DAR-mediated genome cyclization regulates viral replication differently and is under different selection pressures in mammalian and mosquito cells. A more constrained complementarity of the DAR is required in mosquito cells than in mammalian cells. Since the DAR element is stably maintained among mosquito-borne flaviviruses, our findings could provide new information for understanding the role of flavivirus genome cyclization in viral adaptation and RNA evolution in the two hosts.