Production Of Virus Particles Research Articles

The Involvement of Atlastin in Dengue Virus and Wolbachia Infection in Aedes aegypti and Its Regulation by aae-miR-989.

ABSTRACTEndoplasmic reticulum (ER)-shaping atlastin proteins (ATLs) have been demonstrated to play a functional role during flavivirus replication in mammalian cells. For dengue virus (DENV), atlastin is required in the formation of the replication organelles and RNA replication, virion assembly, production of the infectious virus particles, and trafficking or directing the association of vesicle packets with furin. Here, we investigated the involvement of atlastin in DENV replication in the mosquito Aedes aegypti and explored the possibility of its manipulation by the endosymbiotic bacterium Wolbachia to interfere with DENV replication. Results showed the expression of Ae. aegypti atlastin gene (AaATL) was upregulated in DENV-infected Aag2 cells, and its silencing led to reduced DENV replication. Contrary to our assumption that AaATL could be downregulated by Wolbachia, we did not find evidence for that in Wolbachia-infected cell lines, but this was the case in mosquitoes. Further, silencing AaATL did not have any effect on Wolbachia density. Our results also suggest that aae-miR-989 miRNA negatively regulates AaATL. The oversupply of the miRNA mimic led to reduced DENV replication consistent with the positive role of AaATL in DENV replication. Overall, the results favor AaATL’s involvement in DENV replication; however, there is no support that the protein is involved in Wolbachia-mediated DENV inhibition. In addition, the results contribute to discerning further possible overlapping functions of ATLs in mosquitoes and mammalian cells.IMPORTANCE Atlastin is a protein associated with the endoplasmic reticulum and has been shown to play a role in replication of flaviviruses in mammalian cells. This study aimed to investigate the role of mosquito Aedes aegypti atlastin (AaATL) in dengue virus replication and maintenance of Wolbachia, an endosymbiotic bacterium, in the mosquito. Our results suggest that AaATL facilitates dengue virus replication in mosquito cells, considering silencing the gene led to reductions in virus replication and virion production. Further, AaATL was found to be regulated by a mosquito microRNA, aae-miR-989. Despite an effect on dengue virus, AaATL silencing did not affect Wolbachia replication and maintenance in mosquito cells. The results shed light on the role of atlastins in mosquito-pathogen interactions and their overlapping roles in mosquito and mammalian cells.

Tuning Rex rules HTLV-1 pathogenesis.

HTLV-1 is an oncovirus causing ATL and other inflammatory diseases such as HAM/TSP and HU in about 5% of infected individuals. It is also known that HTLV-1-infected cells maintain a disease-free, immortalized, latent state throughout the lifetimes of about 95% of infected individuals. We believe that the stable maintenance of disease-free infected cells in the carrier is an intrinsic characteristic of HTLV-1 that has been acquired during its evolution in the human life cycle. We speculate that the pathogenesis of the virus is ruled by the orchestrated functions of viral proteins. In particular, the regulation of Rex, the conductor of viral replication rate, is expected to be closely related to the viral program in the early active viral replication followed by the stable latency in HTLV-1 infected T cells. HTLV-1 and HIV-1 belong to the family Retroviridae and share the same tropism, e.g., human CD4+ T cells. These viruses show significant similarities in the viral genomic structure and the molecular mechanism of the replication cycle. However, HTLV-1 and HIV-1 infected T cells show different phenotypes, especially in the level of virion production. We speculate that how the activity of HTLV-1 Rex and its counterpart HIV-1 Rev are regulated may be closely related to the properties of respective infected T cells. In this review, we compare various pathological aspects of HTLV-1 and HIV-1. In particular, we investigated the presence or absence of a virally encoded “regulatory valve” for HTLV-1 Rex or HIV-1 Rev to explore its importance in the regulation of viral particle production in infected T cells. Finally, wereaffirm Rex as the key conductor for viral replication and viral pathogenesis based on our recent study on the novel functional aspects of Rex. Since the activity of Rex is closely related to the viral replication rate, we hypothesize that the “regulatory valve” on the Rex activity may have been selectively evolved to achieve the “scenario” with early viral particle production and the subsequent long, stable deep latency in HTLV-1 infected cells.

Rare POLN mutations confer risk for familial nasopharyngeal carcinoma through weakened Epstein-Barr virus lytic replication.

SummaryBackgroundNasopharyngeal carcinoma (NPC) exhibits significant familial aggregation; however, its susceptibility genes are largely unknown. Thus, this study aimed to identify germline mutations that might contribute to the risk of familial NPC, and explore their biological functions.MethodsWhole-exome sequencing was performed in 13 NPC pedigrees with multiple cases. Mutations co-segregated with disease status were further validated in a cohort composed of 563 probands from independent families, 2,953 sporadic cases, and 3,175 healthy controls. Experimental studies were used to explore the functions of susceptibility genes and their disease-related mutations.FindingsThe three rare missense mutations in POLN (DNA polymerase nu) gene, P577L, R303Q, and F545C, were associated with familial NPC risk (5/576 [0·87%] in cases vs. 2/3374 [0·059%] in healthy controls with an adjusted OR of 44·84 [95% CI:3·91-514·34, p = 2·25 × 10−3]). POLN was involved in Epstein-Barr virus (EBV) lytic replication in NPC cells in vitro. POLN promoted viral DNA replication, immediate-early and late lytic gene expression, and progeny viral particle production, ultimately affecting the proliferation of host cells. The three mutations were located in two pivotal functional domains and were predicted to alter the protein stability of POLN in silico. Further assays demonstrated that POLN carrying any of the three mutations displayed reduced protein stability and decreased expression levels, thereby impairing its ability to promote complete EBV lytic replication and facilitate cell survival.InterpretationWe identified a susceptibility gene POLN for familial NPC and elucidated its function.FundingThis study was funded by the National Key Research and Development Program of China (2021YFC2500400); the National Key Research and Development Program of China (2020YFC1316902); the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007); the National Natural Science Foundation of China (81973131); the National Natural Science Foundation of China (82003520); the National Natural Science Foundation of China (81903395).

Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Although the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (∼100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wild-type (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 38% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.

The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.

The Cholesterol Transport Inhibitor U18666A Interferes with Pseudorabies Virus Infection.

Many viruses require the maintenance of lysosomal cholesterol homeostasis for a successful infection; however, the role of lysosomal cholesterol homeostasis in the alphaherpesvirus life cycle is not clear. Here we show that the lysosomal cholesterol transport inhibitor U18666A interferes with the replication of pseudorabies virus (PRV), a member of the alphaherpesvirus subfamily. The treatment with U18666A caused a significant reduction in the production of infectious virus particles. The U18666A treatment was shown to suppress the release of PRV particles. Pretreating PRV virions with U18666A did not affect virus production, whereas pretreating target cells with U18666A led to a substantial reduction in virus yield. Our previous study showed that two cyclodextrin derivatives, 2-hydroxypropyl-β-cyclodextrin (HPβCD) and 2-hydroxypropyl-γ-cyclodextrin (HPγCD), can rescue the cholesterol accumulation defect in primary fibroblasts derived from a Niemann–Pick disease type C (NPC) patient. Here, we demonstrate that treatment with HPβCD or HPγCD not only rescues the U18666A-induced cholesterol accumulation but also rescues the U18666A-induced inhibition of PRV production. Collectively, our data suggest that U18666A interferes with PRV infection via altering cellular functions that are critical for the viral life cycle and may include lysosomal cholesterol homeostasis.

Porcine cytomegalovirus in xenotransplantation: The new frontier in human transplantation?

Porcine cytomegalovirus in xenotransplantation: The new frontier in human transplantation?

Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1.

Borna disease virus 1 (BoDV-1) is a non-segmented, negative-strand RNA virus that is characterized by persistent infection in the nucleus and low production of progeny virions. This feature impedes not only the harvesting of infectious viral particles from infected cells but also the rescue of high titres of recombinant BoDV-1 (rBoDV-1) by reverse genetics. Here, we demonstrate that exogenous expression of both matrix protein (M) and glycoprotein (G), which are constituents of the viral lipid envelope, significantly facilitates the formation of infectious particles and propagation of BoDV-1 without affecting its viral RNA synthesis. Furthermore, simultaneous transfection of M and G expression plasmids with N, P and L helper plasmids by reverse genetics drastically enhances the rescue efficiency of rBoDV-1. On the other hand, we also show that overexpression of M induces obvious cytotoxicity similar to that of other Mononegaviruses. Together with our recent report showing that excess expression of G induces aberrant accumulation of immature G, a potential stimulator of the host innate immune response, it is conceivable that BoDV-1 may suppress excess expression of M and G to reduce the cytopathic effect, thereby leading to maintenance of persistent infection. Our results contribute not only to the establishment of an efficient method to recover high-titre BoDV-1 but also to understanding the unique mechanism of persistent BoDV-1 infection.

EGFR Activation Impairs Antiviral Activity of Interferon Signaling in Brain Microvascular Endothelial Cells During Japanese Encephalitis Virus Infection

The establishment of Japanese encephalitis virus (JEV) infection in brain microvascular endothelial cells (BMECs) is thought to be a critical step to induce viral encephalitis with compromised blood–brain barrier (BBB), and the mechanisms involved in this process are not completely understood. In this study, we found that epidermal growth factor receptor (EGFR) is related to JEV escape from interferon-related host innate immunity based on a STRING analysis of JEV-infected primary human brain microvascular endothelial cells (hBMECs) and mouse brain. At the early phase of the infection processes, JEV induced the phosphorylation of EGFR. In JEV-infected hBMECs, a rapid internalization of EGFR that co-localizes with the endosomal marker EEA1 occurred. Using specific inhibitors to block EGFR, reduced production of viral particles was observed. Similar results were also found in an EGFR-KO hBMEC cell line. Even though the process of viral infection in attachment and entry was not noticeably influenced, the induction of IFNs in EGFR-KO hBMECs was significantly increased, which may account for the decreased viral production. Further investigation demonstrated that EGFR downstream cascade ERK, but not STAT3, was involved in the antiviral effect of IFNs, and a lowered viral yield was observed by utilizing the specific inhibitor of ERK. Taken together, the results revealed that JEV induces EGFR activation, leading to a suppression of interferon signaling and promotion of viral replication, which could provide a potential target for future therapies for the JEV infection.

PML Alternative Splice Products Differentially Regulate HAdV Productive Infection.

ABSTRACTPromyelocytic leukemia nuclear bodies (PML-NBs) were considered to maintain antiviral capacity, as these spherical complexes are antagonized by viruses. Actual work provides evidence, that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Several early HAdV proteins target PML-NBs, such as E4orf3 that promotes redistribution into track-like structures. PML-associated dependency factors that enhance viral gene expression, such as Sp100A remain in the nuclear tracks while restrictive factors, such as Daxx, are inhibited by either proteasomal degradation or relocalization to repress antiviral functions. Here, we did a comprehensive analysis of nuclear PML isoforms during HAdV infection. Our results show cell line specific differences as PML isoforms differentially regulate productive HAdV replication and progeny production. Here, we identified PML-II as a dependency factor that supports viral progeny production, while PML-III and PML-IV suppress viral replication. In contrast, we identified PML-I as a positive regulator and PML-V as a restrictive factor during HAdV infection. Solely PML-VI was shown to repress adenoviral progeny production in both model systems. We showed for the first time, that HAdV can reorganize PML-NBs that contain PML isoforms other then PML-II. Intriguingly, HAdV was not able to fully disrupt PML-NBs composed out of the PML isoforms that inhibit viral replication, while PML-NBs composed out of PML isoforms with beneficial influence on the virus formed tracks in all examined cells. In sum, our findings clearly illustrate the crucial role of PML-track formation in efficient viral replication.IMPORTANCE Actual work provides evidence that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Alternatively spliced PML isoforms I-VII are expressed from one single pml gene containing nine exons and their transcription is tightly controlled and stimulated by interferons and p53. Several early HAdV proteins target PML-NBs, such as E4orf3, promoting redistribution into track-like structures. Our comprehensive studies indicate a diverging role of PML isoforms throughout the course of productive HAdV infection in either stably transformed human lung (H1299) or liver (HepG2) cells, in which we observed a multivalent regulation of HAdV by all six PML isoforms. PML-I and PML-II support HAdV-mediated track formation and efficient formation of viral replication centers, thus promoting HAdV productive infection. Simultaneously, PML-III, -IV,-V, and -VI antagonize viral gene expression and particle production.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR.

Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host’s genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.

EVALUATION OF Murine norovirus (MNV)-1 INFECTION IN PRIMARY MOUSE B CELLS.

Abstract Methods Primary mouse spleen B cells were stimulated or not with LPS (10ug/ml) + IL-4 (10IU/ml) for 48 hours. Two million cells were infected 72 h with MNV-1 at a MOI of 5. The non-structural (NS7) viral protein expression was analyzed in cell lysates by Western blot with a specific anti-NS7 antibody. The supernatant-associated virus in Raw 264.7 cells was determined by paque assay. Results B cells can be infected by MNV-1, even though they do not express the functional receptor CD300LD and CD300LF present in macrophages and Tuft cells. Since MNV-1 can uses CD44 as a receptor in BMDCs, we wanted to investigate if this molecule has a role in MNV-1 entry into B cells. First, we corroborated MNV-1 infection of B cells by evaluating the presence of NS7 by western blot and viral particles production in the supernatant at 72 hpi. To evaluate the role of CD44 in MNV-1 entry into B cells, we blocked the CD44 receptor using two different anti-CD44 monoclonal antibodies, and we found a significant decrease in both the amount of NS7 protein and virus yield, indicating that CD44 plays a role during virus binding/entry into B cells. Since CD44 expression is increased in LPS + IL-4 activated B cells, we wanted to determine if MNV-1 infection could be favored in these conditions. A correlation between the activation of B cells for 48 h and a significant increase of twice of MNV-1 NS7 protein and virus yield production were detected after infection. Conclusion Blocking CD44 correlates with a reduction of MNV-1 protein synthesis and virus yield production, indicating a role of CD44 in MNV-1 binding/entry. The increase of MNV NS7 protein and virus yield production in C57BL/6J spleen B cells activated with LPS + IL-4 that overexpresses CD44 on its surface, also supports this conclusion. CEMR is supported by a Conacyt fellowship, CVU fellowship number: 780860

Two Conserved Phenylalanine Residues in the E1 Fusion Loop of Alphaviruses Are Essential for Viral Infectivity.

Alphaviruses infect cells by a low pH-dependent fusion reaction between viral and host cell membranes that is mediated by the viral E1 glycoprotein. Most reported alphavirus E1 sequences include two phenylalanines (F87 and F95) in the fusion loop, yet the role of these residues in viral infectivity remains to be defined. Following introduction of wild type (WT), E1-F87A, and E1-F95A chikungunya virus (CHIKV) RNA genomes into cells, viral particle production was similar in magnitude. However, CHIKV E1-F87A and E1-F95A virions displayed impaired infectivity compared with WT CHIKV particles. Although WT, E1-F87A, and E1-F95A particles bound cells with similar efficiencies, E1-F87A and E1-F95A particles were unable to undergo fusion and entry into cells. Introduction of an F95A mutation in the E1 fusion loop of Mayaro virus or Venezuelan equine encephalitis virus also resulted in poorly infectious virions. We further tested whether an E1-F87A or E1-F95A mutation could be incorporated into a live-attenuated vaccine strain, CHIKV 181/25, to enhance vaccine safety. Infection of immunocompromised Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice with 181/25E1-F87A or 181/25E1-F95A resulted in 0% mortality, compared with 100% mortality following 181/25 infection. Despite this enhanced attenuation, surviving Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice were protected against virulent virus re-challenge. Moreover, single-dose immunization of WT mice with either 181/25, 181/25E1-F87A, or 181/25E1-F95A elicited CHIKV-specific antibody responses and protected against pathogenic CHIKV challenge. These studies define a critical function for residues E1-F87 and E1-F95 in alphavirus fusion and entry into target cells and suggest that incorporation of these mutations could enhance the safety of live-attenuated alphavirus vaccine candidates. IMPORTANCE Alphaviruses are human pathogens that cause both debilitating acute and chronic musculoskeletal disease and potentially fatal encephalitis. In this study, we determined that two highly conserved phenylalanine residues in the alphavirus E1 glycoprotein are required for fusion of viral and host cell membranes and viral entry into target cells. We further demonstrated that mutation of these phenylalanines results in a substantial loss of viral virulence but not immunogenicity. These data enhance an understanding of the viral determinants of alphavirus entry into host cells and could contribute to the development of new antivirals targeting these conserved phenylalanines or new live-attenuated alphavirus vaccines.

Effects of reduced gag cleavage efficiency on HIV-1 Gag-Pol package

BackgroundHIV-1 pol, which encodes enzymes required for virus replication, is initially translated as a Gag-Pol fusion protein. Gag-Pol is incorporated into virions via interactions with Gag precursor Pr55gag. Protease (PR) embedded in Gag-Pol mediates the proteolytic processing of both Pr55gag and Gag-Pol during or soon after virus particle release from cells. Since efficient Gag-Pol viral incorporation depends on interaction with Pr55gag via its N-terminal Gag domain, the prevention of premature Gag cleavage may alleviate Gag-Pol packaging deficiencies associated with cleavage enhancement from PR.ResultsWe engineered PR cleavage-blocking Gag mutations with the potential to significantly reduce Gag processing efficiency. Such mutations may mitigate the negative effects of enhanced PR activation on virus assembly and Gag-Pol packaging due to an RT dimerization enhancer or leucine zipper dimerization motif. When co-expressed with Pr55gag, we noted that enhanced PR activation resulted in reduced Gag-Pol cis or trans incorporation into Pr55gag particles, regardless of whether or not Gag cleavage sites within Gag-Pol were blocked.ConclusionsOur data suggest that the amount of HIV-1 Gag-Pol or Pol viral incorporation is largely dependent on virus particle production, and that cleavage blocking in the Gag-Pol N-terminal Gag domain does not exert significant impacts on Pol packaging.

Design, Synthesis, and Preclinical Profiling of GSK3739936 (BMS-986180), an Allosteric Inhibitor of HIV-1 Integrase with Broad-Spectrum Activity toward 124/125 Polymorphs.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have garnered special interest because of their novel mechanism of action: they inhibit HIV-1 replication by promoting aberrant integrase multimerization, leading to the production of replication-deficient viral particles. The binding site of ALLINIs is in a well-defined pocket formed at the interface of two integrase monomers that is characterized by conserved residues along with two polymorphic amino acids at residues 124 and 125. The design, synthesis, and optimization of pyridine-based allosteric integrase inhibitors are reported here. Optimization was conducted with a specific emphasis on the inhibition of the 124/125 polymorphs such that the designed compounds showed excellent potency in vitro against majority of the 124/125 variants. In vivo profiling of promising preclinical lead 29 showed that it exhibited a good pharmacokinetic (PK) profile in preclinical species, which resulted in a low predicted human efficacious dose. However, findings in rat toxicology studies precluded further development of 29.

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication.

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leverage proximity proteomics and conduct a small interfering RNA (siRNA) screen to define the functional interactome of EBOV polymerase. As a proof of principle, we validate two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduces viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host interactome of the EBOV polymerase and to illuminate host-dependent regulation of viral RNA synthesis.

Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus.

Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.

Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean-Congo Hemorrhagic Fever Virus.

Removal of genes coding for major parts of capsid (C), premembrane (prM), and envelope (E) proteins on the flavivirus genome aborts the production of infectious virus particles where the remaining genome forms a replicon that retains replicability in host cells. The C-prM-E proteins can also be expressed in trans with the flavivirus replicons to generate single-round infectious replicon virus-like particles (RVPs). In this study, we characterized the use of RVPs based on the Kunjin strain of WNV (WNVKUN) as a putative WNV vaccine candidate. In addition, the WNVKUN C-prM-E genes were substituted with the Crimean–Congo hemorrhagic fever virus (CCHFV) genes encoding the glycoproteins Gn and Gc to generate a WNVKUN replicon expressing the CCHFV proteins. To generate RVPs, the WNVKUN replicon was transfected into a cell line expressing the WNVKUN C-prM-E. Using immunoblotting and immunofluorescence assays, we showed that the replicon can express the CCHFV Gn and Gc proteins and the RVPs can transduce cells to express WNVKUN proteins and the CCHFV Gn and Gc proteins. Our study also revealed that these RVPs have potential as a vaccine platform with low risk of recombination as it infects cells only in one cycle. The immunization of mice with the RVPs resulted in high seroconversion to both WNV E and NS1 but limited seroconversion to CCHFV Gn and Gc proteins. Interestingly, we found that there was enhanced production of WNV E, NS1 antibodies, and neutralizing antibodies by the inclusion of CCHFV Gc and Gn into WNVKUN RVPs. Thus, this study indicates a complementary effect of the CCHFV Gn and Gc proteins on the immunogenicity by WNVKUN RVPs, which may be applied to develop a future vaccine against the WNV.

Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SignificanceIn this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.