Department of Veterinary Medicine Theses and Dissertations
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Item ZIKA VIRUS RECRUITS CELLULAR PROTEINS TO SUPPORT ITS REPLICATION(2024) Chang, Peixi; Zhang, Yanjin YJ; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Zika virus (ZIKV) is a mosquito-borne pathogen with a massive impact on global public health due to its association with severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. The ZIKV epidemic in the Americas in 2015-2016 and its continuing spread in tropical regions have highlighted the urgent need to understand the molecular mechanisms of viral replication to develop effective antiviral strategies. However, many aspects of how ZIKV interacts with host cells remain unclear. This study identifies and characterizes host factors contributing to ZIKV replication. First, karyopherin alpha 6 (KPNA6) contributes to ZIKV replication by interacting with the ZIKV non-structural protein NS2B. Characterization and mutational analyses identified two essential amino acid residues within NS2B that are critical for interacting with KPNA6. The substitution of these two residues of NS2B in an infectious ZIKV cDNA clone resulted in a significant reduction in viral replication, suggesting that the NS2B-KPNA6 interaction plays a vital role in the viral life cycle. Further studies found that KPNA6 contributes to ZIKV RNA synthesis. Mass spectrometry analysis of the KPNA6 interactome showed that KPNA6 interacts with proteins involved in RNA synthesis, suggesting that ZIKV recruits these factors by promoting KPNA6-binding. Second, this study developed an effective method to isolate the ZIKV replication complex, a membranous structure where viral RNA is synthesized. Proteomic analysis of the isolated complex led to identifying numerous host proteins associated with the viral replication machinery. Among these proteins, human replication factor C subunit 2 (RFC2), an accessory factor involved in DNA replication and repair, was discovered to facilitate ZIKV replication, making it a potential target for therapeutic interventions. In conclusion, this study reveals crucial host factors essential for ZIKV infection and replication and provides insights into the ZIKV-cell interactions. These findings offer new possibilities for developing novel antiviral strategies for controlling future viral outbreaks.Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item Development of improved recombinant NDV-vectored vaccines against highly pathogenic avian influenza virus (HPAIV)(2023) Roy Chowdhury, Ishita; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Highly pathogenic avian influenza viruses (HPAIV) are highly contagious and economically devastating poultry pathogens with a documented transmission to humans causing severe human infections with high mortality. Circulation of these viruses is of public health concern as they have the pandemic potential to mutate to increase transmissibility among humans. The diversity of zoonotic influenza viruses causing human infections is alarming and effective vaccination is needed to control these viruses. Influenza viruses particularly with H7 and H5 subtypes of HA can naturally switch to a highly pathogenic phenotype through different mechanisms. Currently available vaccines are not satisfactory as they are mostly inactivated vaccines that require labor-intensive administration methods and provide suboptimal protection of vaccinated birds. Viral vectors offer crucial advantages over traditional vaccines, including induction of outstanding antibody and cytotoxic lymphocyte responses which is important for the control of viral infections. Newcastle Disease virus (NDV) is a promising vaccine vector for HPAIV since it is highly restricted for replication in the respiratory tract of poultry, it can be easily administered, and it induces both local and systemic immune responses. H7 influenza viruses are classified into two major genetic lineages, American and Eurasian. To develop a universal anti-H7 vaccine, we generated NDV vectors expressing chimeric HA sequences covering both North American and Asian isolates. In the first project, we designed NDV-vectored vaccines against HPAI H7N8 infection. The Hemagglutinin (HA) protein of influenza viruses is responsible for virus attachment to host cell and is the major target of the humoral immune response. Accordingly, we developed vaccines against HPAIV by generating recombinant NDV vectored H7 serotype-specific vaccines expressing HA protein. We also evaluated the protective efficacy of these recombinant vaccines against highly virulent H7 challenges in both broiler chickens and turkeys and the results were promising for broiler chickens, but for turkeys the vaccination design and scheme need to be further modified. In the second part of the study, we designed some recombinant NDV-vectored vaccines with an increased level of expression of H5HA antigen. The transcriptional unit of NDV contains a major open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by conserved transcriptional initiation and termination control sequences. Previous studies have shown that the addition of UTRs of P, M, and F genes positively modulated foreign gene expression. Hence, we hypothesized that cognate NDV mRNA UTRs would improve the expression of a protective antigen by an NDV-vectored vaccine. We generated recombinant NDVs where the HA of the HPAIV strain H5N1 is flanked by 5’ and 3’UTRs of NDV genes and determined the growth characteristics of these recombinant viruses, their stability, the level of HA expression and their transcription and translation modulation. Both studies aimed for the advancement of NDV-vectored vaccines emphasizing the fact of better expression of the protective antigen and improved immunogenicity for avian influenza virus considering two important strains of H5 and H7.Item DEVELOPMENT OF AVIAN PARAMYXOVIRUS VECTORED VACCINES(2022) Elbehairy, Mohamed Adel; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Avulaviruses (formerly Avian Paramyxoviruses, APMV) are important pathogens of avian species and have been used as viral vectors for more than two decades. Among all APMVs, Newcastle disease virus (NDV or APMV-1) has been most extensively used as a vaccine vector for protection against avian and animal diseases, and as an oncolytic agent. For poultry vaccination, the preexisting maternal antibodies against NDV can neutralize APMV-1 vectors resulting in vaccination failure. Hence, there is a need to develop new vaccine vectors that would escape neutralization by the maternal antibodies. In the first part of my study, I created a reverse genetics system for Avian paramyxovirus-3 strain Wisconsin (APMV-3 Wisc.) which was proven to be nonpathogenic for day-old chicks and embryonated chicken eggs. The virus was used as a vector to express the enhanced green fluorescent protein (GFP) as a heterologous antigen. The recombinant APMV-3 Wisc. expressing GFP was compared with similarly constructed APMV-1 strain LaSota and APMV-3 strain Netherlands-based vectors for GFP expression and growth kinetics in vitro, and for immunogenicity, safety, and tissue tropism in day-old specific pathogen-free (SPF) chicks. APMV-3 strain Netherlands (APMV-3 Neth.) showed the highest growth rate and GFP expression in chicken fibroblast DF-1 cells, followed by APMV-1 LaSota and APMV-3 Wisconsin. In day-old chicks, APMV-3 Neth. spread to different organs, decreased feed intake and caused stunted growth. APMV-3 Wisc. and APMV-1 LaSota were confined to the respiratory tract and did not induce any pathogenic effects. All three constructs induced seroconversion of the vaccinated chicks for the vector antigens. Thus, the reverse genetics system created in this study for APMV-3 Wisc. allows the development of safe APMV vector antigenically different from NDV that can be used for day-old chicks vaccination. In addition, it provides a tool to study the molecular basis of APMV3 pathogenesis. In the second part of my study, I explored a novel approach for the expression of a foreign gene as an uninterrupted open reading frame (ORF) with a cognate gene of NDV vector. This approach is expected to promote the foreign gene expression stability. Avian influenza virus (AIV) hemagglutinin (HA) protein-coding sequence was fused in-frame with various proteins of NDV vector, with a 2A self-cleaving peptide, a furin cleavage site, or both, placed between the AIV and NDV sequences for separation of the two proteins. Among different constructs tested, we only recovered viable viruses with AIV HA fused C-terminally to the NDV HN gene. These viruses demonstrated a higher expression level of AIV HA than the vector constructed according to a traditional scheme of expressing the transgene as a separate transcriptional unit. Also, they showed increased stability of the transgene expression over multiple passages in embryonated chicken eggs. Our results demonstrate the advantages and limitations of this novel method of foreign gene expression that need to be considered for the development of NDV-based vaccine or therapeutic vectors.Item Identification and functional characterization of the GBF1-controlled network of host proteins supporting enterovirus replication(2022) Moghimi, Seyedehmahsa; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genus Enterovirus of the Picornaviridae family contains many established and emerging pathogens. However, licensed vaccines are currently available only against poliovirus and enterovirus A71. No therapeutics have been officially approved to treat any enterovirus infections, although some are being developed. To find suitable targets for antivirals and control the infections, we need to understand the virus's life cycle better and identify the cellular factors involved in virus infection. Enterovirus genome replication occurs on the unique membranes known as replication organelles (ROs). A Golgi resident protein, GBF1, is recruited to the ROs by a viral protein 3A. GBF1 activates small GTPases Arf, which are critical regulators of the cellular secretory pathway. Here, we investigated the mechanistic details of GBF1-dependent Arf activation during enterovirus replication and characterized the proteome of the ROs in the vicinity of GBF1. We showed that Arf1 appeared to be the first to associate with the ROs, followed by other Arfs. Once activated and recruited to the ROs, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that they do not actively cycle between GTP- and GDP-bound states in infected cells. siRNA depletion studies demonstrated an increased sensitivity of polio replication to inhibition of GBF1 in Arf1-, and to a lesser extent, Arf6-depleted cells, indicating the importance of GBF1-mediated activation of these Arfs for the viral replication. Taking advantage of the GBF1 recruitment to the ROs and GBF1’s essential role in enterovirus replication, we used a GBF1 construct fused to APEX2 peroxidase to explore the proteome of the ROs by proximity biotinylation. Among the proteins biotinylated in infected cells were the known cellular factors recruited to the ROs, including PI4KIII, OSBP, and ACBD3, indicating that these proteins are localized close to GBF1. Among the viral proteins, the intermediate products of the polyprotein processing were overrepresented, suggesting that GBF1 is localized close to the sites of active polyprotein processing. About 85% of the proteins identified by MS have not been previously associated with enterovirus infection. Gene ontology analysis revealed a significant enrichment of RNA binding and mRNA metabolic processes, suggesting a close localization of GBF1 to the RNA replication complexes. siRNA knockdown functional analysis of the selected proteins showed the recruitment of both proviral and antiviral factors to the ROs. Collectively, our work revealed important details about the involvement of Arfs in the replication process, introduced a highly efficient system to investigate the proteome of the enterovirus ROs, and provided novel data about the protein composition of the GBF1-enriched environment in the replication sites.Item Development of avian paramyxovirus 3 as a vaccine vector against infectious bursal disease in one-day-old specific pathogen free chickens(2021) Varghese, Berin Parambethu; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACTInfectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease affecting young chickens, resulting in substantial economic losses to the poultry industry worldwide. Although strict hygienic measures and various vaccination strategies have been adopted, IBD remains a major problem for the poultry industry. The economic significance of this disease is exhibited in two ways. First, the disease can cause high morbidity and mortality. Second, severe prolonged immunosuppression of chickens when infected at an early age leads to susceptibility to other diseases and vaccination failure. Therefore, chicks should be protected from early infection, usually achieved by vaccinating breeder flocks and active immunization of the newly hatched chicks. Vaccines include live-attenuated, viral vectored recombinant, subunit, and inactivated vaccines currently available to control infectious bursal disease (IBD). The major flaws of available vaccines are the reversion to virulence, the generation of new variant viruses, inability to replicate in the presence of maternal antibodies, lack of proper delivery systems, recombination and integration into the host genome, and inadequate systemic immune response. To overcome these obstacles, we have evaluated avian paramyxoviruses (APMVs) as vaccine vectors to generate vaccines against IBD. In the present study, we constructed recombinant Newcastle disease virus (NDV) strain LaSota (rLaSota/VP2) and recombinant avian paramyxovirus-3 (APMV-3) strain Netherlands expressing VP2 protein (rAPMV-3/VP2), the immunogenic protein of IBDV, and to evaluate their protective efficacies following immunization of one-day-old specific pathogen-free (SPF) chicks. Our results showed that both recombinant viruses stably express the VP2 protein, and their in vitro growth characteristics were almost similar to their respective parental viruses. Immunization of one-day-old SPF chicks demonstrated that rAPMV-3/VP2 protein elicited IBDV specific neutralizing antibodies and provided complete protection against the IBDV STC challenge. In addition, the rAPMV-3/VP2 protects chickens from clinical signs, gross lesions, and histopathology even at lower vaccine doses. Moreover, rAPMV-3/VP2 provides slightly better protection than the commercial vaccine from histopathology lesion against IBD at four weeks of age. This study suggests that recombinant APMV-3 expressing VP2 protein could be used as a potential vaccine against IBD in field conditions where maternal antibodies exist.Item HEPATITIS E VIRUS MODULATES HOST FACTORS TO GENERATE A CONDUCIVE ENVIRONMENT FOR REPLICATION(2020) lin, shaoli; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV infection mainly presents as acute and self-limiting hepatitis in young adults. However, it can be exacerbated to fulminant hepatitis in HEV-infected pregnant women, resulting in up to 30% case fatality. Besides, chronic HEV infection with rapid progression in immunocompromised patients has been a challenge in many countries since it was reported years ago. HEV infection is zoonotic, and human HEV strains are grouped into four major genotypes in the genus Orthohepevirus A, the family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. Due to the lack of an effective cell culture system and a proper animal model, HEV biology, virus-cell interactions, and pathogenesis are understudied. HEV is known to inhibit the innate immune response by targeting type I interferon (IFN) signaling pathway via its ORF1 products. Nevertheless, it remains largely unknown how the virus manipulates host factors to facilitate its replication. The objective of these studies was to elucidate the mechanism of HEV manipulation of host factors to generate a conducive environment for replication. Our results show that the capsid protein of HEV inhibits the IFN production to dampen the antiviral response through its N-terminal arginine-rich motif. In addition to the impairment of innate immunity, HEV proliferation requires the presence of other host factors: DDX3, an RNA helicase, and oxysterol-binding protein (OSBP), a lipid transporter. The knockdown of these two factors led to a significant reduction of HEV replication, whereas the reconstitution of these two genes restores the HEV proliferation level. The capsid protein was found to interact with the C-terminal domain of DDX3. The HEV helicase was shown to interact with OSBP and block its translocation to the Golgi apparatus. These results indicate that HEV employs multiple strategies including blocking antiviral response and recruiting host factors for its invasion and proliferation. Our data provide insights into the HEV-cell interactions and may facilitate the development of novel antiviral strategies.Item AVIAN PARAMYXOVIRUS-VECTORED VACCINES AGAINST INFECTIOUS BRONCHITIS VIRUS AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS(2019) Shirvani, Edris; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines. Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV. Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts. Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions. In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.Item Antagonizing JAK-STAT signaling by porcine reproductive and respiratory syndrome virus(2018) Yang, Liping; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by numerous cytokines. JAK-STAT pathways involve in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of their significance in immune response, they are often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and severe respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One possible reason for the weak protective immune response is that PRRSV interferes with innate immunity and modulates cytokine signaling, including JAK-STAT pathways. The objective of this project was to elucidate the mechanisms of PRRSV interference with JAK-STAT2 and JAK-STAT3 signaling. This study demonstrates that PRRSV antagonizes interferon (IFN)-activated JAK-STAT2 signaling and oncostatin M (OSM)-activated JAK-STAT3 pathway via inducing STAT2 and STAT3 degradation. Mechanistically, PRRSV non-structural protein 11 (nsp11) and nsp5 induce the degradation of STAT2 and STAT3, respectively, via the ubiquitin-proteasome pathway. Notably, PRRSV manipulates karyopherin alpha 6 (KPNA6), an importin that is responsible for STAT3 nuclear translocation in the JAK-STAT signaling, to facilitate viral replication. Knockdown of KPNA6 expression led to significant reduction in PRRSV replication. These data demonstrate that PRRSV interferes with different JAK-STAT pathways to evade host antiviral response while harnessing cellular factors for its own replication. These findings provide new insights into PRRSV-cell interactions and its molecular pathogenesis in interference with the host immune response, and facilitate the development of novel antiviral therapeutics.Item VIRAL IMMUNE EVASION OF FCRN FUNCTIONS(2018) LIU, XIAOYANG; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human Cytomegalovirus (HCMV) is known to evade host immunity, allowing it to persistently infect humans. Although the strategies of HCMV to evade cellular immunity is well studied, there is limited understanding on how HCMV antagonizes humoral immunity. The neonatal Fc receptor (FcRn), an MHC class I-related FcγR, plays a critical role in IgG-mediated humoral immunity. Through screening the HCMV proteome, we discovered that US11 specifically captured FcRn in both virally-infected and US11-expressing cells. US11 selectively inhibited the assembly of FcRn with β2m, impaired FcRn IgG binding capacity and blocked FcRn trafficking to the endosome by retention of FcRn in ER. Furthermore, US11 recruited Derlin-1 and E3 ubiquitin ligase TMEM129, to induce degradation of FcRn in US11+ or HCMV-infected cells. This complex led to the dislocation of FcRn from the ER to the cytosol and facilitated its degradation in an ubiquitination and proteasome-dependent manner. The cytosolic interaction between FcRn and Derlin-1 was shown necessary for degrading FcRn. FcRn is widely expressed in most cell susceptible to HCMV infection, including epithelial, endothelial and macrophage. Our data showed that either HCMV infection or recombinant US11 expression significantly inhibited human IgG transcytosis across polarized human primary intestinal epithelial Caco-2 cells, Vascular endothelial HMEC-1 cells and placental trophoblast BeWo cells, and facilitated considerable IgG degradation inside endothelial HMEC-1 cells. Hence, our results show that HCMV exploits the Derlin-1/TMEM129 pathway through US11 to disable FcRn, revealing a novel strategy for viral evasion from antibody mediated-immunity. We also studied whether HCMV viral FcγRs (gp34 and gp68) and US11 work together to facilitate IgG degradation. HCMV vFcγRs has been reported to internalize IgG via endocytosis. Interestingly, we found that in acidic pH (6.0) condition, the IgG binding capacity of gp34 was largely impaired while the IgG binding capacity of gp68 had minimal change. Consequently, in the presence of FcRn, gp34 did not enhance IgG degradation whereas gp68 significantly promoted the IgG degradation. Furthermore, the presence of US11 induced more gp34 and gp68-mediated IgG degradation in FcRn+ cells.
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