ResearchPad - viral-replication Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The influenza replication blocking inhibitor LASAG does not sensitize human epithelial cells for bacterial infections]]> Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection.

<![CDATA[Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1]]> Promyelocytic leukemia (PML) bodies are liquid droplet-like structures organized by the eponymous PML proteins in the nuclei of our cells. PML bodies have been implicated in the antiviral host cell response to infection. Consequently, viruses have evolved mechanisms that target the proteins composing PML bodies. Immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies produced by the human cytomegalovirus, one of eight human herpesviruses. Previous work suggested that the interaction between IE1 and PML and the consequent disruption of PML bodies serves a critical role in viral replication by counteracting the cellular antiviral response. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be unstable. We systematically screened for stable IE1 variants and identified a mutant protein selectively defective for PML interaction. Unexpectedly, the IE1 mutant supported viral replication almost as efficiently as the wild-type protein. Moreover, lower instead of higher (as expected) levels of antiviral gene expression were observed with the mutant compared to the wild-type. These results suggest that disruption of PML bodies is linked to the induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

<![CDATA[Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein]]>

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.

<![CDATA[In vitro and in silico multidimensional modeling of oncolytic tumor virotherapy dynamics]]>

Tumor therapy with replication competent viruses is an exciting approach to cancer eradication where viruses are engineered to specifically infect, replicate, spread and kill tumor cells. The outcome of tumor virotherapy is complex due to the variable interactions between the cancer cell and virus populations as well as the immune response. Oncolytic viruses are highly efficient in killing tumor cells in vitro, especially in a 2D monolayer of tumor cells, their efficiency is significantly lower in a 3D environment, both in vitro and in vivo. This indicates that the spatial dimension may have a major influence on the dynamics of virus spread. We study the dynamic behavior of a spatially explicit computational model of tumor and virus interactions using a combination of in vitro 2D and 3D experimental studies to inform the models. We determine the number of nearest neighbor tumor cells in 2D (median = 6) and 3D tumor spheroids (median = 16) and how this influences virus spread and the outcome of therapy. The parameter range leading to tumor eradication is small and even harder to achieve in 3D. The lower efficiency in 3D exists despite the presence of many more adjacent cells in the 3D environment that results in a shorter time to reach equilibrium. The mean field mathematical models generally used to describe tumor virotherapy appear to provide an overoptimistic view of the outcomes of therapy. Three dimensional space provides a significant barrier to efficient and complete virus spread within tumors and needs to be explicitly taken into account for virus optimization to achieve the desired outcome of therapy.

<![CDATA[Comparative fitness of West Nile virus isolated during California epidemics]]>

West Nile virus (WNV) has been circulating in California since its first detection in 2003, causing repeated outbreaks affecting public, wildlife and veterinary health. Epidemics of WNV are difficult to predict due to the multitude of factors influencing transmission dynamics among avian and mosquito hosts. Typically, high levels of WNV amplification are required for outbreaks to occur, and therefore associated viral strains may exhibit enhanced virulence and mortality in competent bird species resulting in increased mosquito infection prevalence. In our previous study, most WNV isolates made from California during 2007–08 showed increased fitness when competed in House Finches (HOFI, Haemorhous mexicanus) and Culex tarsalis Coquillett mosquitoes against COAV997-5nt, a genetically marked recombinant virus derived from a 2003 California strain. Herein, we evaluated the competitive fitness of WNV strains isolated during California epidemics in 2004, 2005, 2007, 2011 and 2012 against COAV997-5nt. These outbreak isolates did not produce elevated mortality in HOFIs, but replicated more efficiently than did COAV997-5nt based on quantification of WNV RNA copies in sera, thereby demonstrating increased competitive fitness. Oral co-infections in Cx. tarsalis resulted in similar virus-specific infection and transmission rates, indicating that outbreak isolates did not have a fitness advantage over COAV997-5nt. Collectively, WNV isolates from outbreaks demonstrated relatively greater avian, but not vector, replicative fitness compared to COAV997-5nt, similar to previously characterized non-outbreak isolates of WNV. Our results indicated that ecological rather than viral factors may facilitate WNV amplification to outbreak levels, but monitoring viral phenotypes through competitive fitness studies may provide insight into altered replication and transmission potential among emerging WNV strains.

<![CDATA[Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo]]>

Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir

<![CDATA[Vector competence of biting midges and mosquitoes for Shuni virus]]>


Shuni virus (SHUV) is an orthobunyavirus that belongs to the Simbu serogroup. SHUV was isolated from diverse species of domesticated animals and wildlife, and is associated with neurological disease, abortions, and congenital malformations. Recently, SHUV caused outbreaks among ruminants in Israel, representing the first incursions outside the African continent. The isolation of SHUV from a febrile child in Nigeria and seroprevalence among veterinarians in South Africa suggests that the virus may have zoonotic potential as well. The high pathogenicity, extremely broad tropism, potential transmission via both biting midges and mosquitoes, and zoonotic features of SHUV require further investigation. This is important to accurately determine the risk for animal and human health, and to facilitate preparations for potential epidemics. To gain first insight into the potential involvement of biting midges and mosquitoes in SHUV transmission we have investigated the ability of SHUV to infect two species of laboratory-colonised biting midges and two species of mosquitoes.

Methodology/Principal findings

Culicoides nubeculosus, C. sonorensis, Culex pipiens pipiens, and Aedes aegypti were orally exposed to SHUV by providing an infectious blood meal. Biting midges showed high infection rates of approximately 40%-60%, whereas infection rates of mosquitoes were only 0–2%. Moreover, successful dissemination in both species of biting midges and no evidence for transmission by orally exposed mosquitoes was found.


The results of this study suggest that different species of Culicoides midges are efficient in SHUV transmission, while the involvement of mosquitoes has not been supported.

<![CDATA[Infectious complications and NK cell depletion following daratumumab treatment of Multiple Myeloma]]>

Treatment with Daratumumab (Dara), a monoclonal anti-CD38 antibody of IgG1 subtype, is effective in patients with multiple myeloma (MM). However, Dara also impairs the cellular immunity, which in turn may lead to higher susceptibility to infections. The exact link between immune impairment and infectious complications is unclear. In this study, we report that nine out of 23 patients (39%) with progressive MM had infectious complications after Dara treatment. Five of these patients had viral infections, two developed with bacterial infections and two with both bacterial and viral infections. Two of the viral infections were exogenous, i.e. acute respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), while five consisted of reactivations, i.e. one herpes simplex (HSV), 1 varicella-zoster (VZV) and three cytomegalovirus (CMV). Infections were solely seen in patients with partial response or worse. Assessment of circulating lymphocytes indicated a selective depletion of NK cells and viral reactivation after Dara treatment, however this finding does not exclude the multiple components of viral immune-surveillance that may get disabled during this monoclonal treatment in this patient cohort. These results suggest that the use of antiviral and antibacterial prophylaxis and screening of the patients should be considered.

<![CDATA[Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription]]>

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

<![CDATA[Mechanistic framework predicts drug-class specific utility of antiretrovirals for HIV prophylaxis]]>

Currently, there is no effective vaccine to halt HIV transmission. However, pre-exposure prophylaxis (PrEP) with the drug combination Truvada can substantially decrease HIV transmission in individuals at risk. Despite its benefits, Truvada-based PrEP is expensive and needs to be taken once-daily, which often leads to inadequate adherence and incomplete protection. These deficits may be overcome by next-generation PrEP regimen, including currently investigated long-acting formulations, or patent-expired drugs. However, poor translatability of animal- and ex vivo/in vitro experiments, and the necessity to conduct long-term (several years) human trials involving considerable sample sizes (N>1000 individuals) are major obstacles to rationalize drug-candidate selection. We developed a prophylaxis modelling tool that mechanistically considers the mode-of-action of all available drugs. We used the tool to screen antivirals for their prophylactic utility and identify lower bound effective concentrations that can guide dose selection in PrEP trials. While in vitro measurable drug potency usually guides PrEP trial design, we found that it may over-predict PrEP potency for all drug classes except reverse transcriptase inhibitors. While most drugs displayed graded concentration-prophylaxis profiles, protease inhibitors tended to switch between none- and complete protection. While several treatment-approved drugs could be ruled out as PrEP candidates based on lack-of-prophylactic efficacy, darunavir, efavirenz, nevirapine, etravirine and rilpivirine could more potently prevent infection than existing PrEP regimen (Truvada). Notably, some drugs from this candidate set are patent-expired and currently neglected for PrEP repurposing. A next step is to further trim this candidate set by ruling out compounds with ominous safety profiles, to assess different administration schemes in silico and to test the remaining candidates in human trials.

<![CDATA[Virus load and clinical features during the acute phase of Chikungunya infection in children]]>


Chikungunya virus (CHIKV) infection is a long known mosquito-borne disease that is associated with severe morbidity, characterized by fever, headache, rashes, joint pain, and myalgia. It is believed that virus load has relation with severity of clinical features.


We performed this study to assess the relationship between virus load and clinical features in children during the acute phase of CHIKV infection, in order to draw insights for better-informed treatment.

Study design

Between June 1, 2009, and May 31, 2010, 338 patients with fever and susceptive to CHIKV during first 4 days of illness were prospectively enrolled from Karnataka Institute of Medical Sciences, Hubli in our hospital based cross sectional observational study. Sybr green quantitative reverse transcription polymerase chain reaction was performed to estimate the virus load.


Quantitative RT-PCR was positive for CHIKV in 54 patients. The median copy number of CHIKV was 1.3x 108 copies/ml (1.7x105-9.9x109 copies/ml). Among the observed clinical features, a statistically significant difference in log mean virus load was found between patients with and without myalgia (log mean 7.50 vs 8.34, P = 0.01).


Patients with myalgia had lower virus load and those without myalgia had a higher virus load.

<![CDATA[Treatment of HIV among tuberculosis patients: A replication study of timing of antiretroviral therapy for HIV-1-associated tuberculosis]]>

Co-diagnosis of HIV and tuberculosis presents a treatment dilemma. Starting both treatments at the same time can cause a flood of immune response called immune reconstitution inflammatory syndrome (IRIS) which can be lethal. But, how long to delay HIV treatment is less understood. In 2011, based on the conclusions of three separate studies, WHO recommended starting HIV treatment earlier for those with later HIV disease progression. This paper conducts a replication study of one of the three studies, by Havlir and colleagues. Using their publicly available data, we were able to replicate most of the results presented in the original paper. In our measurement and estimation analyses we use different estimation techniques to assess the robustness of the results. We find that adjusting for loss to follow-up does not affect the main results of the paper. However, an ANCOVA estimation and an instrumental variable model weaken the main result of the paper of better outcomes with early HIV treatment only for those who are sicker, reducing significance from the 5% to the 10% level. A change-point analysis also detects no changes in effect by timing of HIV treatment initiation or different thresholds of CD4 count for the primary outcome. This result suggests that the choice of start time for HIV treatment initiation should be based on other factors including potential drug interactions, overlapping side effects, a high pill burden and severity of illness rather than CD4 threshold and preset timeframes. While we caution against overgeneralizing, the result of this replication is aligned with more recent studies that show no evidence that early initiation of HIV treatment reduces mortality for any patients.

<![CDATA[Toll-like receptor 3 regulates Zika virus infection and associated host inflammatory response in primary human astrocytes]]>

The connection between Zika virus (ZIKV) and neurodevelopmental defects is widely recognized, although the mechanisms underlying the infectivity and pathology in primary human glial cells are poorly understood. Here we show that three isolated strains of ZIKV, an African strain MR766 (Uganda) and two closely related Asian strains R103451 (Honduras) and PRVABC59 (Puerto Rico) productively infect primary human astrocytes, although Asian strains showed a higher infectivity rate and increased cell death when compared to the African strain. Inhibition of AXL receptor significantly attenuated viral entry of MR766 and PRVABC59 and to a lesser extend R103451, suggesting an important role of TAM receptors in ZIKV cell entry, irrespective of lineage. Infection by PRVABC59 elicited the highest release of inflammatory molecules, with a 8-fold increase in the release of RANTES, 10-fold increase in secretion of IP-10 secretion and a 12-fold increase in IFN-β secretion when compared to un-infected human astrocytes. Minor changes in the release of several growth factors, endoplasmic reticulum (ER)-stress response factors and the transcription factor, NF-κB were detected with the Asian strains, while significant increases in FOXO6, MAPK10 and JNK were detected with the African strain. Activation of the autophagy pathway was evident with increased expression of the autophagy related proteins Beclin1, LC3B and p62/SQSTM1 with all three strains of ZIKV. Pharmacological inhibition of the autophagy pathway and genetic inhibition of the Beclin1 showed minimal effects on ZIKV replication. The expression of toll-like receptor 3 (TLR3) was significantly increased with all three strains of ZIKV; pharmacological and genetic inhibition of TLR3 caused a decrease in viral titers and in viral-induced inflammatory response in infected astrocytes. We conclude that TLR3 plays a vital role in both ZIKV replication and viral-induced inflammatory responses, irrespective of the strains, while the autophagy protein Beclin1 influences host inflammatory responses.

<![CDATA[Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli]]>

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

<![CDATA[The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing]]>

Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great need for animal models that accurately mimic PV infection in humans for testing therapeutic strategies against human papillomaviruses (HPVs). In this study, we present data that demonstrate that in terms of gene expression during initial viral DNA amplification, Macaca fascicularis PV (MfPV) types 5 and 8 appear to be similar to mucosal oncogenic HPVs, while MfPV1 (isolated from skin) resembles most high-risk cutaneous beta HPVs (HPV5). Similarities were also observed in replication properties during the initial amplification phase of the MfPV genomes. We demonstrate that high-risk mucosal HPV-specific inhibitors target the transient replication of the MfPV8 genomes, which indicates that similar pathways are used by the high-risk HPVs and MfPVs during their genome replication. Taking all into account, we propose that Macaca fascicularis may serve as a highly relevant model for preclinical tests designed to evaluate therapeutic strategies against HPV-associated lesions.

<![CDATA[Fibrillar structures induced by a plant reovirus target mitochondria to activate typical apoptotic response and promote viral infection in insect vectors]]>

Numerous plant viruses that cause significant agricultural problems are persistently transmitted by insect vectors. We wanted to see if apoptosis was involved in viral infection process in the vector. We found that a plant reovirus (rice gall dwarf virus, RGDV) induced typical apoptotic response during viral replication in the leafhopper vector and cultured vector cells, as demonstrated by mitochondrial degeneration and membrane potential decrease. Fibrillar structures formed by nonstructural protein Pns11 of RGDV targeted the outer membrane of mitochondria, likely by interaction with an apoptosis-related mitochondrial protein in virus-infected leafhopper cells or nonvector insect cells. Such association of virus-induced fibrillar structures with mitochondria clearly led to mitochondrial degeneration and membrane potential decrease, suggesting that RGDV Pns11 was the inducer of apoptotic response in insect vectors. A caspase inhibitor treatment and knockdown of caspase gene expression using RNA interference each reduced apoptosis and viral accumulation, while the knockdown of gene expression for the inhibitor of apoptosis protein improved apoptosis and viral accumulation. Thus, RGDV exploited caspase-dependent apoptotic response to promote viral infection in insect vectors. For the first time, we directly confirmed that a nonstructural protein encoded by a persistent plant virus can induce the typical apoptotic response to benefit viral transmission by insect vectors.

<![CDATA[HIV-1 infection increases microRNAs that inhibit Dicer1, HRB and HIV-EP2, thereby reducing viral replication]]>

HIV-1 is the causative agent of AIDS (Autoimmune Deficiency Syndrome). HIV-1 infection results in systemic CD4+ T cell depletion, thereby impairing cell-mediated immunity. MicroRNAs are short (~22 nucleotides long), endogenous single-stranded RNA molecules that regulate gene expression by binding to the 3' untranslated regions (3' UTR) of mRNA transcripts. The relation between HIV-1 infection and human miRNA expression profile has been previously investigated, and studies have shown that the virus can alter miRNA expression and vice versa. Here, we broaden the understanding of the HIV-1 infection process, and show that miRNA-186, 210 and 222 are up-regulated following HIV-1 infection of human Sup-T1 cells. As a result, the host miRNA target genes: Dicer1 (Double-Stranded RNA-Specific Endoribonuclease), HRB (HIV-1 Rev-binding protein) and HIV-EP2 (Human Immunodeficiency Virus Type I Enhancer Binding Protein 2), are down-regulated. Moreover, testing the miRNA-gene anti- correlation on the Jurkat and the HeLa-MAGI cell lines demonstrated the ability of the miRNAs to down-regulate viral expression as well. To conclude, we found that human miR-186, 210 and 222 directly regulate the human genes Dicer1, HRB and HIV-EP2, thus may be filling key roles during HIV-1 replication and miRNA biogenesis. This finding may contribute to the development of new therapeutic strategies.

<![CDATA[Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus]]>

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

<![CDATA[Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity]]>

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.

<![CDATA[Unexpected lessons from the neglected: How defective viral genomes became important again]]> ]]>