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1.
Mario Mietzsch Ariana Jose Paul Chipman Nilakshee Bhattacharya Nadia Daneshparvar Robert McKenna Mavis Agbandje-McKenna 《Viruses》2021,13(1)
The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting. 相似文献
2.
Renuk Lakshmanan Mario Mietzsch Alberto Jimenez Ybargollin Paul Chipman Xiaofeng Fu Jianming Qiu Maria Sderlund-Venermo Robert McKenna 《Viruses》2022,14(10)
Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10–30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics. 相似文献
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4.
The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus–host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid–host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid–host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics. 相似文献
5.
Penetration of the viral genome into a host cell nucleus is critical for initiation of viral replication for most DNA viruses and a few RNA viruses. For herpesviruses, viral DNA ejection into a nucleus occurs when the capsid docks at the nuclear pore complex (NPC) basket with the correct orientation of the unique capsid portal vertex. It has been shown that capsid vertex-specific component (CVSC) proteins, which are located at the twelve vertices of the human herpes simplex virus type 1 (HSV-1) capsid, interact with nucleoporins (Nups) of NPCs. However, it remained unclear whether CVSC proteins determine capsid-to-NPC binding. Furthermore, it has been speculated that terminal DNA adjacent to the portal complex of DNA-filled C-capsids forms a structural motif with the portal cap (which retains DNA in the capsid), which mediates capsid-NPC binding. We demonstrate that terminal viral DNA adjacent to the portal proteins does not present a structural element required for capsid-NPC binding. Our data also show that level of CVSC proteins on the HSV-1 capsid affects level of NPC binding. To elucidate the capsid-binding process, we use an isolated, reconstituted cell nucleus system that recapitulates capsid-nucleus binding in vivo without interference from trafficking kinetics of capsids moving toward the nucleus. This allows binding of non-infectious capsid maturation intermediates with varying levels of vertex-specific components. This experimental system provides a platform for investigating virus–host interaction at the nuclear membrane. 相似文献
6.
Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and mammalian orthoreoviruses was revealed. However, the functions of the aquareovirus genome-encoded proteins are poorly understood. We investigated the molecular characteristics of the outer capsid proteins, namely, VP5 and VP7, of grass carp reovirus (GCRV). The peptides VP5 and VP7 were determined using in-gel tryptic digestion and mass spectrometry. Recovered peptides represented 76% and 66% of the full-length VP5 and VP7 sequences, respectively. Significantly, two-lysine acetylation, as well as two-serine and two-threonine phosphorylation modifications, were first revealed in VP5. We found that the initial amino acid in VP5 was Pro43, suggesting that a lower amount of VP5 remained uncleaved in virions at the autocleavage site (Asn42-Pro43). Further biochemical evidence showed that the cleaved VP5N/VP5C conformation was the major constituent of the particles. Moreover, early cleavage fragments of VP7 and enhanced infectivity were detected after limited tryptic digestion of GCRV, indicating that stepwise VP7 cleavage is essential for VP5 conformational rearrangement. Our results provide insights into the roles of posttranslational modifications in VP5 and its association with VP7 in the viral life cycle. 相似文献
7.
Zhu Yang Michael Reeves Jun Ye Phong Trang Li Zhu Jingxue Sheng Yu Wang Ke Zen Jianguo Wu Fenyong Liu 《Viruses》2015,7(7):3345-3360
An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. 相似文献
8.
Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus. 相似文献
9.
To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. 相似文献
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Qinfang Sun Ronald M. Levy Karen A. Kirby Zhengqiang Wang Stefan G. Sarafianos Nanjie Deng 《Viruses》2021,13(5)
While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207. 相似文献
12.
Zhong L Li B Mah CS Govindasamy L Agbandje-McKenna M Cooper M Herzog RW Zolotukhin I Warrington KH Weigel-Van Aken KA Hobbs JA Zolotukhin S Muzyczka N Srivastava A 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(22):7827-7832
Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy. 相似文献
13.
Philippe A. Gallay Roger G. Ptak Michael D. Bobardt Jean-Maurice Dumont Grégoire Vuagniaux Brigitte Rosenwirth 《Viruses》2013,5(3):981-997
DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent. 相似文献
14.
Daniel J. Goetschius Samantha R. Hartmann Lindsey J. Organtini Heather Callaway Kai Huang Carol M. Bator Robert E. Ashley Alexander M. Makhov James F. Conway Colin R. Parrish Susan L. Hafenstein 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(23)
Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab–virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab–virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.Canine parvovirus (CPV) emerged as a host range variant virus in the mid-1970s, subsequently causing a pandemic of disease in dogs during 1978 (1, 2). Since that time, multiple variants have emerged with additional mutations in the viral capsid (3, 4). Extensive genetic and biochemical studies have shown that specific mutations displayed on or near the capsid surface alter binding to the host receptor, transferrin receptor type-1 (TfR). Since the specific host ranges of canine and feline parvoviruses are primarily controlled by the ability of the virus to bind TfR, changes in the binding site alter the ability of the virus to infect different hosts (3, 5, 6).The virus capsid is highly antigenic, and an infection elicits many different host antibodies, which recognize specific structures on the surface of the virus that are primarily displayed as conformational epitopes. Most antibodies efficiently neutralize virus as IgGs, whereas they vary in their neutralization abilities when tested as Fabs (7, 8). In a number of cases, selection of antibody escape mutations by antibodies also selects for host range variation in the viruses, and conversely, selection for host range variation alters the antigenic structure recognized by specific antibodies (6, 9, 10). Although some of these changes appear to result from overlap of the receptor and antibody binding sites, it is still not clear how different selections operate in the natural evolution of the viruses. Understanding the mechanisms of host recognition and the dynamics of the binding by antibodies and receptors would provide insight into the connections between antigenic and host range variation, enabling us to predict the ability of a given virus capsid to change hosts or to escape host immunity. The coordinated overlap between antibody and receptor binding has also been seen in other viruses, including SARS-CoV-2 and influenza viruses (11–13).CPV has a small, 26-nm diameter, T = 1 icosahedral capsid that packages a single-stranded DNA (ssDNA) genome of about 5,000 bases. The capsid shell is composed of VP2 (∼90%) and VP1 (∼10%), which are generated by differential messenger RNA splicing events so that the entire sequence of VP2 is also contained within VP1. Both proteins fold into the same eight-stranded, antiparallel β-barrel structure, where the β-strands are connected by loops that make up the surface features of the capsid. A raised region known as the threefold spike surrounds each icosahedral threefold axis and contains most of the antigenic structures recognized by different antibodies (14, 15). MAb 14 is a mouse monoclonal antibody generated against CPV capsids that has particularly interesting properties. MAb 14 binding, hemagglutination inhibition properties, and neutralization are all virus-strain specific, and it bound with significantly higher affinity to CPV capsids than to the closely related but host range variant–virus that infected cats, feline panleukopenia virus (FPV) (16–18). The virus-specific binding of MAb is controlled by the capsid surface residue 93, which is Lys in FPV and Asn in CPV (19–21). In addition to antibody recognition, residue 93 also controls canine host range, since Asn93 allows binding to canine TfR and infection of canine cells, whereas Lys93 in the equivalent position on the FPV capsid prevents both of these processes (20).Despite the central role of antibodies in protecting animals against virus infections and allowing recovery from disease, in many cases, we still lack a detailed understanding of epitope characteristics, the dynamics of binding processes, and the viral neutralization mechanisms. Previous X-ray crystallography and cryo-electron microscopy (cryo-EM) structures include the Fab of MAb 14 (Fab 14), CPV and FPV capsids, and a Fab 14–CPV capsid complex at moderate resolution of 12.5 Å (Protein Data Bank [PDB] IDs: 2CAS, 1C8F, 3IY0, and 3GK8) (18, 22, 23). Crystal structures of Fab 14, CPV, and FPV fitted into the cryo-EM map of Fab–virus complex have allowed us to predict protein interactions in the binding interface (18). Although this was the most rigorous approach at the time, the resulting pseudoatomic structure based on the fitting did not explain why variation in residue 93 controlled Fab binding, identify likely mechanisms of antibody neutralization, or explain how the minor changes in the site also affect TfR binding. Recent technological advances in cryo-EM now allow us to solve Fab–virus structures at high enough resolution to build atomic models directly into the density map for identifying interactions unambiguously.The binding and occupancy of Fabs on the CPV capsid have also been determined previously using charge detection mass spectrometry, which revealed that some of the tested monoclonal antibody–derived Fabs, including Fab 14, could fully occupy all 60 epitopes of the capsid but with some differences in the kinetics of attachment (24). Incubating with excess Fab molecules to occupy all icosahedrally equivalent sites on capsids has long been the preferred cryo-EM structural approach since this allows icosahedral symmetry averaging to be imposed during the reconstruction process for maximizing resolution (18, 25, 26). However, there are few other studies confirming the occupancies of Fabs on viral capsids, and the larger IgG likely does not saturate the entire surface of the capsid so that an undersaturated capsid (with fewer than 60 bound Fab in the case of parvoviruses) would more closely mimic the physiologically relevant complexes. Solving such an asymmetric structure at atomic resolution is now possible due to advances in cryo-EM and the reconstruction approaches.Here, we define an atomic model of Fab 14 bound to the capsid of CPV based on cryo-EM of the complex and examine the functional mechanisms that affect binding by testing antibody mutants. Of the two data sets used to reconstruct Fab–virus complex maps, one had close to complete occupancy of the 60 capsid epitopes, whereas the other had an average of 10 Fabs bound per capsid. These data were used initially to solve the icosahedrally averaged structures of fully Fab-occupied and partially Fab-occupied complexes to resolutions of 3.2 and 2.3 Å, respectively. An asymmetric, partially Fab-occupied virus map calculated with local reconstruction approaches attained 2.4-Å global resolution and revealed the Fab-occupied and unoccupied sites on the same virus capsid. These structures allowed unambiguous identification of residues and side chains involved in the Fab–virus binding interface and also revealed local conformational changes in the antibody binding site induced by Fab binding. The partial occupancy of the capsids by Fab provided an opportunity to develop an innovative algorithm to test for complementarity of Fab binding to different positions on the capsid. Notably, it was the asymmetric approach and not the traditional icosahedrally averaged reconstruction that revealed the details of antibody binding. 相似文献
15.
Lucas Y. H. Goh Jody Hobson-Peters Natalie A. Prow Kelly Baker Thisun B. H. Piyasena Carmel T. Taylor Ashok Rana Marcus L. Hastie Jeff J. Gorman Roy A. Hall 《Viruses》2015,7(6):2943-2964
Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP. 相似文献
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17.
Shuai Qi Wangjun Fu Jinyan Fan Li Zhang Binyang Zheng Kang Wang Xiangxi Wang Ling Zhu Xinjian Li Yuxia Zhang 《Viruses》2022,14(11)
Echovirus 3 (E3), a serotype of human enterovirus B (HEV-B), causes severe diseases in infants. Here, we determined the structures of E3 with a monoclonal antibody (MAb) 6D10 by cryo-EM to comprehensively understand the specificities and the immunological characteristic of this serotype. The solved cryo-EM structures of the F-, A-, and E-particles of E3 bound with 6D10 revealed the structural features of the virus–antibody interface. Importantly, the structures of E-particles bound with 6D10 revealed for the first time the nature of the C-terminus of VP1 for HEV-Bs at the structural level. The highly immunogenic nature of this region in the E-particles provides new strategies for vaccine development for HEV-Bs. 相似文献
18.
Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry
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Fokine A Leiman PG Shneider MM Ahvazi B Boeshans KM Steven AC Black LW Mesyanzhinov VV Rossmann MG 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(20):7163-7168
Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor. 相似文献
19.
Rajkumar Lalji Sahani Raquel Diana-Rivero Sanjeev Kumar V. Vernekar Lei Wang Haijuan Du Huanchun Zhang Andres Emanuelli Castaner Mary C. Casey Karen A. Kirby Philip R. Tedbury Jiashu Xie Stefan G. Sarafianos Zhengqiang Wang 《Viruses》2021,13(3)
Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 μM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism. 相似文献
20.
Adrian R. Wilkie Mayuri Sharma Margaret Coughlin Jean M. Pesola Maria Ericsson Jessica L. Lawler Rosio Fernandez Donald M. Coen 《Viruses》2022,14(3)
After herpesviruses encapsidate their genomes in replication compartments (RCs) within the nuclear interior, capsids migrate to the inner nuclear membrane (INM) for nuclear egress. For human cytomegalovirus (HCMV), capsid migration depends at least in part on nuclear myosin Va. It has been reported for certain herpesviruses that the nucleoplasmic subunit of the viral nuclear egress complex (NEC) is important for this migration. To address whether this is true for HCMV, we used mass spectrometry and multiple other methods to investigate associations among the HCMV NEC nucleoplasmic subunit, UL53, myosin Va, major capsid protein, and/or capsids. We also generated complementing cells to derive and test HCMV mutants null for UL53 or the INM NEC subunit, UL50, for their importance for these associations and, using electron microscopy, for intranuclear distribution of capsids. We found modest associations among the proteins tested, which were enhanced in the absence of UL50. However, we found no role for UL53 in the interactions of myosin Va with capsids or the percentage of capsids outside RC-like inclusions in the nucleus. Thus, UL53 associates somewhat with myosin Va and capsids, but, contrary to reports regarding its homologs in other herpesviruses, is not important for migration of capsids towards the INM. 相似文献