Viral Factor Research Articles

Unraveling factors responsible for pathogenic differences in Lassa virus strains.

Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a severe hemorrhagic disease with potential for lethal outcomes. Apart from acute symptoms, LF survivors often endure long-term complications, notably hearing loss, which significantly impacts their quality of life and socioeconomic status in endemic regions of West Africa. Classified as a Risk Group 4 agent, LASV poses a substantial public health threat in affected areas. Our laboratory previously developed a novel lethal guinea pig model of LF utilizing the clinical isolate LASV strain LF2384. However, the specific pathogenic factors underlying LF2384 infection in guinea pigs remained elusive. In this study, we aimed to elucidate the differences in the immunological response induced by LF2384 and LF2350, another LASV isolate from a non-lethal LF case within the same outbreak. Through comprehensive immunological gene profiling, we compared the expression kinetics of key genes in guinea pigs infected with LASV LF2384 and LF2350. Our analysis revealed differential expression patterns for several immunological genes, including CD94, CD19-2, CD23, IL-7, and CIITA, during LF2384 and LF2350 infection. Moreover, through the generation of recombinant LASVs, we sought to identify the specific viral genes responsible for the observed pathogenic differences between LF2384 and LF2350. Our investigations pinpointed the L protein as a crucial determinant of pathogenicity in guinea pigs infected with LASV LF2384.

Selection and characterization of aptamers targeting the Vif-CBFβ-ELOB-ELOC-CUL5 complex.

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. Whilst recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention.

Virological Profile of Asthma Exacerbation in Children: A Hospital-Based Retrospective Study.

Introduction Viruses are the most common triggering factors for asthma exacerbation during the autumn and winter seasons. Viruses,such as influenza A and rhinovirus, play a major role in the occurrence of severe exacerbation ofasthma. This association between viral infection and asthma exacerbation in children is a result of the antiviral response of the immune system and various anti-inflammatory phenomena. In this work, we aimed to identify the virological profile of asthma exacerbation in children and analyze the correlation between viral infection type and the severity of exacerbation. Materials and methods This retrospective study was conducted from January 2016 to January 2024. The study included children hospitalized for asthma exacerbation associated with signs of viral-like respiratory infection with positive virological testing by multiplex real-time polymerase chain reaction or rapid test in the case of influenza A or respiratory syncytial virus (RSV). Data analysis was performed with Microsoft Excel and SPSS software using a previously established data collection sheet Results Thirty cases were collected for the study period. The mean age of the patients was 4 years and 8 months, with a male-to-female ratio of 3.3. Eighteen patients were known to have asthma, of which nine had uncontrolled asthma, and exacerbation was inaugural in 12 patients. Viral shedding was found in 14 patients. A viral agent was found in all patients, with coinfection of two or more viruses in three patients. The viruses found were influenza A (18 cases), coupled rhinovirus/enterovirus (eight cases), RSV (eight cases), human metapneumovirus (three patients), and parainfluenza type IV in only one inaugural patient. Asthma exacerbation was severe in 20 patients, moderate in eight patients, and two patients had severe acute asthma requiring intensive care management. We noted a higher frequency of severe exacerbation among those with an influenza A viral infection. All patients with RSV infection exhibited moderate exacerbation. No other significant correlation between asthma severity and other types of viruses was found. Conclusions Our results demonstrate the major role played by viruses in triggering asthma exacerbation, primarily influenza virus, followed by enterovirus, rhinovirus, RSV, and metapneumovirus. Larger-scale studies should be carried out to establish a more complete virological profile and further investigate the viral factor in the management of asthma in children.

The varicella-zoster virus ORF16 protein promotes both the nuclear transport and the protein abundance of the viral DNA polymerase subunit ORF28

Although all herpesviruses utilize a highly conserved replication machinery to amplify their viral genomes, different members may have unique strategies to modulate the assembly of their replication components. Herein, we characterize the subcellular localization of seven essential replication proteins of varicella-zoster virus (VZV) and show that several viral replication enzymes such as the DNA polymerase subunit ORF28, when expressed alone, are localized in the cytoplasm. The nuclear import of ORF28 can be mediated by the viral DNA polymerase processivity factor ORF16. Besides, ORF16 could markedly enhance the protein abundance of ORF28. Noteworthily, an ORF16 mutant that is defective in nuclear transport still retained the ability to enhance ORF28 abundance. The low abundance of ORF28 in transfected cells was due to its rapid degradation mediated by the ubiquitin-proteasome system. We additionally reveal that radicicol, an inhibitor of the chaperone Hsp90, could disrupt the interaction between ORF16 and ORF28, thereby affecting the nuclear entry and protein abundance of ORF28. Collectively, our findings imply that the cytoplasmic retention and rapid degradation of ORF28 may be a key regulatory mechanism for VZV to prevent untimely viral DNA replication, and suggest that Hsp90 is required for the interaction between ORF16 and ORF28.

ORF virus causes tumor-promoting inflammation in sheep and goats.

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.

The Molecular Characterization of the Cyprinid herpesvirus 3 (CyHV-3) ORF24 Protein and its effect on the expression of immune genes (in vitro)

Cyprinid herpesvirus 3 (CyHV-3), known as koi herpesvirus (KHV), is highly contagious and lethal. In this study, we aimed to characterize the ORF24-encoding protein of CyHV-3, investigate its sub-cellular localization, and determine its impact on the expression of immune factors through in vitro experiments. The results showed that the CyHV-3 ORF24 protein comprises 579 amino acids. Interestingly, multiple comparisons with homologous proteins from three carp herpesvirus origins showed no significant similarity. The fluorescence localization experiment showed that the green fluorescence signal, representing the protein pEGFP-ORF24, was primarily diffused in the cytoplasm. Notably, the overexpression of ORF24 effectively suppressed the expression of immune factors in both CCO (Channel catfish ovary) and FHM (Fathead minnow muscle cell line) cells. Bioinformatic analysis indicated that the CyHV-3 ORF24 gene exhibited significant differences from the corresponding genes in the other two carp herpesviruses. This suggests its unique functional role in the evolutionary context. Moreover, our findings demonstrated that overexpression of CyHV-3 ORF24 can effectively inhibit the expression of immune factors, underscoring its crucial role as a viral immune escape factor. These results provide further insights into the immune function of the CyHV-3 ORF24 protein and offer a theoretical foundation for developing new vaccines against CyHV-3 virus infections.

PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

The P2 nucleic acid binding protein of Sugarcane bacilliform virus is a viral pathogenic factor.

Saccharum spp. is the primary source of sugar and plays a significant role in global renewable bioenergy. Sugarcane bacilliform virus (SCBV) is one of the most important viruses infecting sugarcane, causing severe yield losses and quality degradation. It is of great significance to reveal the pathogenesis of SCBV and resistance breeding. However, little is known about the viral virulence factors or RNA silencing suppressors and the molecular mechanism of pathogenesis. To systematically investigate the functions of the unknown protein P2 encoded by SCBV ORF2. Phylogenetic analysis was implemented to infer the evolutionary relationship between the P2 of SCBV and other badnaviruses. The precise subcellular localization of P2 was verified in the transient infiltrated Nicotiana benthamiana epidermal mesophyll cells and protoplasts using the Laser scanning confocal microscope (LSCM). The post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) RNA silencing suppressor activity of P2 was analyzed, respectively. Furthermore, restriction digestion and RT-qPCR assays were conducted to verify the probable mechanism of P2 on repressing DNA methylation. To explore the pathogenicity of P2, a potato virus X-based viral vector was used to heterologously express SCBV P2 and the consequent H2O2 accumulation was detected by the 3,3'-diaminobenzidine (DAB) staining method. Phylogenetic analysis shows that SCBV has no obvious sequence similarity and low genetic relatedness to Badnavirus and Tungrovirus representatives. LSCM studies show that P2 is localized in both the cytoplasm and nucleus. Moreover, P2 is shown to be a suppressor of PTGS and TGS, which can not only repress ssRNA-induced gene silencing but also disrupt the host RNA-directed DNA methylation (RdDM) pathway. In addition, P2 can trigger an oxidative burst and cause typical hypersensitive-like response (HLR) necrosis in systemic leaves of N. benthamiana when expressed by PVX. Overall, our results laid a foundation for deciphering the molecular mechanism of SCBV pathogenesis and made progress for resistance breeding.

Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

Inhibition of the Alternative Complement Pathway May Cause Secretion of Factor B, Enabling an Early Detection of Pancreatic Cancer.

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity

PurposeCervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood.MethodsTo investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation.ResultsIn precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells.ConclusionIn aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes.

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids.

HIV-1 Vif protein sequence variations in South African people living with HIV and their influence on Vif-APOBEC3G interaction

PurposeDespite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions.MethodsSanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity.ResultsThe findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups.ConclusionsThese results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.

Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

Generation and Retention of Defective RNA3 from Cucumber Mosaic Virus and Relevance of the 2b Protein to Generation of the Subviral RNA

A defective RNA3 (D3Yα) of strain Y of cucumber mosaic virus (CMV-Y) was examined on host-specific maintenance, experimental conditions, and a viral factor required for its generation in plants. D3Yα was stably maintained in cucumber but not in tomato plants for 28 days post inoculation (dpi). D3Yα was generated in <i>Nicotiana tabacum</i> or <i>N. benthamiana</i> after prolonged infection in the second and the third passages, but not in plants of <i>N. benthamiana</i> grown at low temperature at 28 dpi or infected with CMV-Y mutant that had the 2b gene deleted. Collectively, we suggest that generation and retention of D3Yα depends on potential host plants and experimental conditions, and that the 2b protein has a role for facilitation of generation of D3Yα.

Methionine gamma lyase fused with S3 domain VGF forms octamers and adheres to tumor cells via binding to EGFR

Methods for targeting enzymes exhibiting anticancer properties, such as methionine γ-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 ― MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Herpesvirus lytic infection-induced mitophagy via viral interferon regulatory factor 1.

Viral control of mitochondria via mitophagy has a dampening effect on mitochondrion-mediated innate immune responses. We previously found that human herpesvirus 8 (HHV-8) could activate mitophagy via its lytic gene product vIRF-1 (viral interferon regulatory factor 1). Mechanistically, we previously demonstrated that vIRF-1 interacts with the mitophagic proteins BNIP3L (BCL2 interacting protein 3 like) and TUFM (Tu translation elongation factor, mitochondrial). Despite these significant findings, however, the precise molecular mechanisms underlying vIRF-1-activated mitophagy, particularly with core components of the autophagy machinery, remained to be fully elucidated. We recently reported that vIRF-1 binds preferentially and directly to GABARAPL1 (GABA type A receptor associated protein like 1) in a noncanonical manner, and this interaction is essential for virus-productive replication. Furthermore, we found that BNIP3L is a crucial factor that promotes vIRF-1 oligomerization and associated mitophagy activation, including GABARAPL1 interaction with vIRF-1 and TUFM dimerization. Together, our findings deepen our understanding of lytic infection-induced mitophagy and provide the key protein-protein interactions involved in vIRF-1-mediated mitophagy.

KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

HPV16 E7 protein antagonizes TNF-α-induced apoptosis of cervical cancer cells via Daxx/JNK pathway

Human papillomavirus (HPV) E7 protein as an important viral factor was involved in the progression of cervical cancer by mediating the cellular signaling pathways. Daxx (Death domain-associated protein) can interact with a variety of proteins to affect the viral infection process. However, the interaction and its related function between HPV16 E7 and Daxx have not been adequately investigated. Here, it was found that HPV16 E7 can interact with Daxx in HeLa or C33A cells by co-immunoprecipitation. HPV16 E7 protein treatment can up-regulate Daxx protein expression, while the interference in Daxx expression and the agonists for JNK can both reduce the antagonistic effects of HPV16 E7 on TNF-α-induced apoptosis, suggesting that Daxx/JNK pathway may be involved in the anti-apoptotic activity of HPV16 E7.