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1.
Cell ; 181(5): 1046-1061.e6, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32392465

RESUMO

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.


Assuntos
Vírus Gigantes/genética , Vírus Gigantes/metabolismo , Vírus Gigantes/fisiologia , Capsídeo/metabolismo , Vírus de DNA/genética , Genoma Viral/genética , Proteômica/métodos , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Viroses/genética , Vírus/genética
2.
J Virol ; 97(12): e0142023, 2023 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-37991366

RESUMO

This satellite symposium was focused on the molecular arms race between bacteria and their predators, the bacteriophages: who's the friend and who's the foe? This Gem recounts highlights of the talks and presents food for thought and additional reflections on the current state of the field.


Assuntos
Bactérias , Bacteriófagos , Interações entre Hospedeiro e Microrganismos , Bactérias/metabolismo , Bactérias/virologia , Bacteriófagos/metabolismo , Bacteriófagos/patogenicidade
3.
J Virol ; 96(16): e0092922, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-35894604

RESUMO

The first critical step in a virus's infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable coevolution and host range switching or expansion. Bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) of S. flexneri and then binds outer membrane protein (Omp) A or OmpC. This phage infects serotype Y strains but can also form small, turbid plaques on serotype 2a2; turbid plaques appear translucent rather than transparent, indicating greater survival of bacteria. Reduced plating efficiency further suggested inefficient infection. To examine the interactions between Sf6 and this alternate host, phages were experimentally evolved using mixed populations of S. flexneri serotypes Y and 2a2. The recovered mutants could infect serotype 2a2 with greater efficiency than the ancestral Sf6, forming clear plaques on both serotypes. All mutations mapped to two distinct regions of the receptor-binding tailspike protein: (i) adjacent to the LPS binding site near the N terminus; and (ii) at the distal, C-terminal tip of the protein. Although we anticipated interactions between the Sf6 tailspike and 2a2 O-antigen to be weak, LPS of this serotype appears to inhibit infection through strong binding of particles, effectively removing them from the environment. The mutations of the evolved strains reduce the inhibitory effect by either reducing electrostatic interactions with the O-antigen or increasing reliance on the Omp secondary receptors. IMPORTANCE Viruses depend on host cells to propagate themselves. In mixed populations and communities of host cells, finding these susceptible host cells may have to be balanced with avoiding nonhost cells. Alternatively, being able to infect new cell types can increase the fitness of the virus. Many bacterial viruses use a two-step process to identify their hosts, binding first to an LPS receptor and then to a host protein. For Shigella virus Sf6, the tailspike protein was previously known to bind the LPS receptor. Genetic data from this work imply the tailspike also binds to the protein receptor. By experimentally evolving Sf6, we also show that point mutations in this protein can dramatically affect the binding of one or both receptors. This may provide Sf6 flexibility in identifying host cells and the ability to rapidly alter its host range under selective pressure.


Assuntos
Bacteriófagos/genética , Glicosídeo Hidrolases/genética , Mutação Puntual , Shigella flexneri/virologia , Proteínas da Cauda Viral/genética , Especificidade de Hospedeiro , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Antígenos O/química , Antígenos O/genética , Antígenos O/metabolismo
4.
J Bacteriol ; 203(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33753469

RESUMO

The instability of Shigella genomes has been described, but how this instability causes phenotypic differences within the Shigella flexneri species is largely unknown and likely variable. We describe herein the genome of S. flexneri strain PE577, originally a clinical isolate, which exhibits several phenotypic differences compared to the model strain 2457T. Like many previously described strains of S. flexneri, PE577 lacks discernible, functional CRISPR and restriction-modification systems. Its phenotypic differences when compared to 2457T include lower transformation efficiency, higher oxygen sensitivity, altered carbon metabolism, and greater susceptibility to a wide variety of lytic bacteriophage isolates. Since relatively few Shigella phages have been isolated on 2457T or the previously characterized strain M90T, developing a more universal model strain for isolating and studying Shigella phages is critical to understanding both phages and phage-host interactions. In addition to phage biology, the genome sequence of PE577 was used to generate and test hypotheses of how pseudogenes in this strain-whether interrupted by degraded prophages, transposases, frameshifts, or point mutations-have led to metabolic rewiring compared to the model strain 2457T. Results indicate that PE577 can utilise the less-efficient pyruvate oxidase/acetyl-CoA synthetase (PoxB/Acs) pathway to produce acetyl-CoA, while strain 2457T cannot due to a nonsense mutation in acs, rendering it a pseudogene in this strain. Both strains also utilize pyruvate-formate lyase to oxidize formate but cannot survive with this pathway alone, possibly because a component of the formate-hydrogen lyase (fdhF) is a pseudogene in both strains.Importance Shigella causes millions of dysentery cases worldwide, primarily affecting children under five years old. Despite active research in developing vaccines and new antibiotics, relatively little is known about the variation of physiology or metabolism across multiple isolates. In this work, we investigate two strains of S. flexneri that share 98.9% genetic identity but exhibit drastic differences in metabolism, ultimately affecting the growth of the two strains. Results suggest additional strains within the S. flexneri species utilize different metabolic pathways to process pyruvate. Metabolic differences between these closely-related isolates suggest an even wider variety of differences in growth across S. flexneri and Shigella in general. Exploring this variation further may assist the development or application of vaccines and therapeutics to combat Shigella infections.

5.
Mol Microbiol ; 108(3): 288-305, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29470858

RESUMO

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.


Assuntos
Bacteriófago P22/genética , Lisogenia/genética , Salmonella typhimurium/genética , Bacteriófago P22/patogenicidade , Elementos de DNA Transponíveis/genética , Deleção de Genes , Testes Genéticos/métodos , Lisogenia/fisiologia , Mutação , Salmonella/genética , Fagos de Salmonella/patogenicidade , Transdução Genética
6.
J Virol ; 92(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29437962

RESUMO

In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella-infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica, and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella, our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities.IMPORTANCEShigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella-infecting bacteriophages have been described. By specifically looking for Shigella-infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on "nontraditional" laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages.


Assuntos
Bacteriófagos , Surtos de Doenças , Disenteria Bacilar/epidemiologia , Escherichia coli/virologia , Shigella flexneri/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/metabolismo , Disenteria Bacilar/virologia , Feminino , Humanos , Masculino
7.
J Bacteriol ; 198(23): 3209-3219, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27645387

RESUMO

Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTXϕ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains of Acinetobacter baumannii These CRAϕ-like A. baumannii prophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. IMPORTANCE: We observed the emergence of a novel filamentous phage (CRAϕ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAϕ reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRAϕ is the Vibrio cholerae CTXϕ phage. Variants of CRAϕ are found in the genomes of Acinetobacter baumannii strains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.


Assuntos
Acinetobacter baumannii/virologia , Inovirus/isolamento & purificação , Prófagos/isolamento & purificação , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Genoma Viral , Inovirus/classificação , Inovirus/genética , Inovirus/fisiologia , Prófagos/classificação , Prófagos/genética , Prófagos/fisiologia
8.
J Virol ; 89(2): 1182-94, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25378500

RESUMO

UNLABELLED: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. IMPORTANCE: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.


Assuntos
Giardia lamblia/virologia , Giardiavirus/isolamento & purificação , Giardiavirus/ultraestrutura , Vírion/ultraestrutura , Microscopia Crioeletrônica , Imageamento Tridimensional
9.
Appl Environ Microbiol ; 82(16): 4982-93, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27287319

RESUMO

UNLABELLED: This paper examines the recovery of the enteric adenovirus human adenovirus 40 (HAdV 40) by cross-flow ultrafiltration and interprets recovery values in terms of physicochemical interactions of virions during sample concentration. Prior to ultrafiltration, membranes were either blocked by exposure to calf serum (CS) or coated with a polyelectrolyte multilayer (PEM). HAdV 40 is a hydrophobic virus with a point of zero charge between pH 4.0 and pH 4.3. In accordance with predictions from the extended Derjaguin-Landau-Verwey-Overbeek theory, the preelution recovery of HAdV (rpre) from deionized water was higher with PEM-coated membranes (rpre (PEM) = 74.8% ± 9.7%) than with CS-blocked membranes (rpre (CS) = 54.1% ± 6.2%). With either membrane type, the total virion recovery after elution (rpost) was high for both deionized water (rpost (PEM) = 99.5% ± 6.6% and rpost (CS) = 98.8% ± 7.7%) and tap water (rpost (PEM) = 89% ± 15% and rpost (CS) = 93.7% ± 6.9%). The nearly 100% recoveries suggest that the polyanion (sodium polyphosphate) and surfactant (Tween 80) in the eluent disrupt electrostatic and hydrophobic interactions between the virion and the membrane. Addition of EDTA to the eluent greatly improved the elution efficacy (rpost (CS) = 88.6% ± 4.3% and rpost (PEM) = 87.0% ± 6.9%) with surface water, even when the organic carbon concentration in the water was high (9.4 ± 0.1 mg/liter). EDTA likely disrupts cation bridging between virions and particles in the feed water matrix or the fouling layer on the membrane surface. For complex water matrices, the eluent composition is the most important factor for achieving high virion recovery. IMPORTANCE: Herein we present the results of a comprehensive physicochemical characterization of HAdV 40, an important human pathogen. The data on HAdV 40 surface properties enabled rigorous modeling to gain an understanding of the energetics of virion-virion and virion-filter interactions. Cross-flow filtration for concentration and recovery of HAdV 40 was evaluated, with postelution recoveries from ultrapure water (99%), tap water (∼91%), and high-carbon-content surface water (∼84%) being demonstrated. These results are significant because of the very low adenovirus recoveries that have been reported, to date, for other methods. The recovery data were interpreted in terms of specific interactions, and the eluent composition was designed accordingly to maximize HAdV 40 recovery.


Assuntos
Adenovírus Humanos/isolamento & purificação , Água Potável/virologia , Água Doce/virologia , Ultrafiltração , Interações Hidrofóbicas e Hidrofílicas , Membranas Artificiais , Polieletrólitos , Polímeros/metabolismo , Polifosfatos/metabolismo , Polissorbatos/metabolismo , Compostos de Sódio/metabolismo , Tensoativos/metabolismo , Ultrafiltração/instrumentação , Vírion/isolamento & purificação
10.
Mol Microbiol ; 92(1): 47-60, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24673644

RESUMO

Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófagos/crescimento & desenvolvimento , Lipopolissacarídeos/metabolismo , Shigella flexneri/genética , Shigella flexneri/virologia , Proteínas da Membrana Bacteriana Externa/genética , Bacteriófagos/ultraestrutura , Tomografia com Microscopia Eletrônica , Genoma Viral , Microscopia de Fluorescência , Mutação , Shigella flexneri/metabolismo , Vírion/fisiologia
11.
Structure ; 32(1): 24-34.e4, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-37909043

RESUMO

There is a paucity of high-resolution structures of phages infecting Shigella, a human pathogen and a serious threat to global health. HRP29 is a Shigella podophage belonging to the Autographivirinae family, and has very low sequence identity to other known phages. Here, we resolved the structure of the entire HRP29 virion by cryo-EM. Phage HRP29 has a highly unusual tail that is a fusion of a T7-like tail tube and P22-like tailspikes mediated by interactions from a novel tailspike adaptor protein. Understanding phage tail structures is critical as they mediate hosts interactions. Furthermore, we show that the HRP29 capsid is stabilized by two novel, and essential decoration proteins, gp47 and gp48. Only one high resolution structure is currently available for Shigella podophages. The presence of a hybrid tail and an adapter protein suggests that it may be a product of horizontal gene transfer, and may be prevalent in other phages.


Assuntos
Bacteriófagos , Shigella , Humanos , Microscopia Crioeletrônica , Bacteriófagos/química , Shigella/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Proteínas da Cauda Viral/química
12.
Elife ; 122024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38206122

RESUMO

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.


Assuntos
Acetofenonas , Bacteriófagos , Vibrio cholerae , Capsídeo , Proteínas do Capsídeo , Bacteriófagos/genética , Escherichia coli
13.
bioRxiv ; 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36909475

RESUMO

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, PLE, remodels the capsid it has been predicted to steal from the phage ICP1 (1). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like-particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold, that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (2). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

14.
J Virol ; 85(15): 7483-95, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21593159

RESUMO

Baboon reovirus (BRV) is a member of the fusogenic subgroup of orthoreoviruses. Unlike most other members of its genus, BRV lacks S-segment coding sequences for the outer fiber protein that binds to cell surface receptors. It shares this lack with aquareoviruses, which constitute a related genus and are also fusogenic. We used electron cryomicroscopy and three-dimensional image reconstruction to determine the BRV virion structure at 9.0-Å resolution. The results show that BRV lacks a protruding fiber at its icosahedral 5-fold axes or elsewhere. The results also show that BRV is like nonfusogenic mammalian and fusogenic avian orthoreoviruses in having 150 copies of the core clamp protein, not 120 as in aquareoviruses. On the other hand, there are no hub-and-spoke complexes attributable to the outer shell protein in the P2 and P3 solvent channels of BRV, which makes BRV like fusogenic avian orthoreoviruses and aquareoviruses but unlike nonfusogenic mammalian orthoreoviruses. The outermost "flap" domains of the BRV core turret protein appear capable of conformational variability within the virion, a trait previously unseen among other ortho- and aquareoviruses. New cDNA sequence determinations for the BRV L1 and M2 genome segments, encoding the core turret and outer shell proteins, were helpful for interpreting the structural features of those proteins. Based on these findings, we conclude that the evolution of ortho- and aquareoviruses has included a series of discrete gains or losses of particular components, several of which cross taxonomic boundaries. Gain or loss of adhesion fibers is one of several common themes in double-stranded RNA virus evolution.


Assuntos
Orthoreovirus/química , Vírion/química , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Microscopia Crioeletrônica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Orthoreovirus/fisiologia , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Células Vero , Vírion/genética , Vírion/ultraestrutura
15.
Biochim Biophys Acta Biomembr ; 1864(7): 183920, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35358430

RESUMO

Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops.


Assuntos
Proteínas de Bactérias , Bacteriófagos , Porinas , Shigella , Proteínas de Bactérias/genética , Bacteriófagos/patogenicidade , Porinas/genética , Shigella/genética , Shigella/virologia , Virulência
16.
Nat Microbiol ; 7(8): 1210-1220, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35817890

RESUMO

Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1.


Assuntos
Bacteriófagos , Cólera , Vibrio cholerae , Bacteriófagos/genética , Cólera/microbiologia , Toxina da Cólera , Ilhas Genômicas , Humanos , Vibrio cholerae/genética
17.
Mol Microbiol ; 77(6): 1568-82, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20659287

RESUMO

We have investigated determinants of polyhead formation in bacteriophage P22 in order to understand the molecular mechanism by which coat protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in coat protein at position 170, which is located in the ß-hinge. In vivo scaffolding protein does not correct polyhead assembly by F170A or F170K coat proteins, but does for F170L. All F170 variants bind scaffolding protein more weakly than wild-type as observed by affinity chromatography with scaffolding protein-agarose and scaffolding protein shell re-entry experiments. Electron cryo-microscopy and three-dimensional image reconstructions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the coat subunits relative to wild-type. This was confirmed by limited proteolysis and protein sequencing, which showed increased protection of the A-domain. Our data support the conclusion that the decrease in flexibility of the A-domain leads to crowding of the subunits at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct coat protein interactions with scaffolding protein and maintenance of sufficient coat protein flexibility are crucial for proper P22 assembly. The coat protein ß-hinge region is the major determinant for both features.


Assuntos
Bacteriófago P22/química , Proteínas do Capsídeo/química , Montagem de Vírus , Substituição de Aminoácidos , Bacteriófago P22/genética , Bacteriófago P22/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína
18.
J Virol ; 84(24): 12945-57, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20861247

RESUMO

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (ßB to ßI) ß-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.


Assuntos
Proteínas do Capsídeo/química , Dependovirus/química , Dependovirus/metabolismo , Polissacarídeos/metabolismo , Vírion/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Dependovirus/classificação , Humanos , Modelos Moleculares , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
J Virol ; 84(12): 5880-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20375175

RESUMO

Human bocavirus (HBoV) was recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) on the basis of genomic similarity to bovine parvovirus and canine minute virus. HBoV has been implicated in respiratory tract infections and gastroenteric disease in children worldwide, yet despite numerous epidemiological reports, there has been limited biochemical and molecular characterization of the virus. Reported here is the three-dimensional structure of recombinant HBoV capsids, assembled from viral protein 2 (VP2), at 7.9-A resolution as determined by cryo-electron microscopy and image reconstruction. A pseudo-atomic model of HBoV VP2 was derived from sequence alignment analysis and knowledge of the crystal structure of human parvovirus B19 (genus Erythrovirus). Comparison of the HBoV capsid structure to that of parvoviruses from five separate genera demonstrates strong conservation of a beta-barrel core domain and an alpha-helix, from which emanate several loops of various lengths and conformations, yielding a unique surface topology that differs from the three already described for this family. The highly conserved core is consistent with observations for other single-stranded DNA viruses, and variable surface loops have been shown to confer the host-specific tropism and the diverse antigenic properties of this family.


Assuntos
Capsídeo/química , Parvoviridae/química , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Humanos , Parvoviridae/genética , Parvoviridae/metabolismo , Parvoviridae/ultraestrutura , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Appl Sci (Basel) ; 11(5)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33898062

RESUMO

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn-Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism.

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