RESUMEN

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Asunto(s)

Bacteriófago phi X 174 , Proteínas de la Cápside , ADN de Cadena Simple , ADN Viral , Proteínas de Unión al ADN , Evolución Molecular , Empaquetamiento del Genoma Viral , Bacteriófago phi X 174/química , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/crecimiento & desarrollo , Bacteriófago phi X 174/metabolismo , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Secuencia Conservada , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Aptitud Genética , Mutación , Fenotipo , Moldes Genéticos , Virión/química , Virión/genética , Virión/crecimiento & desarrollo , Virión/metabolismo

RESUMEN

Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this "host-induced" unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, α3 and ST-1. Despite ∼90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from coinfected cells did not display the phenotype. Thus, mutant isolation required populations generated in environments with low multiplicities of infection (MOI), a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.IMPORTANCE To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts, culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low-MOI environments. However, in laboratory settings, viral propagation includes high-MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Last, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.

Asunto(s)

Proteínas de la Cápside/metabolismo , Escherichia coli/virología , Genes Recesivos , Interacciones Huésped-Patógeno/genética , Mutación , Virión , Ensamble de Virus , Secuencia de Aminoácidos , Bacteriófago phi X 174 , Proteínas de la Cápside/genética , Especificidad del Huésped , Microvirus/clasificación , Microvirus/genética , Fenotipo

RESUMEN

Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

Asunto(s)

Bacteriófago phi X 174/fisiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Mutación , Ensamble de Virus , Sustitución de Aminoácidos , Bacteriófago phi X 174/genética , Proteínas de la Cápside/química , Modelos Moleculares , Mutación Missense , Fenotipo , Unión Proteica , Conformación Proteica , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo

RESUMEN

The influence of miRNAs on the host-pathogen environment is largely unknown and under intensive investigation. Whether produced by the pathogen or by the host cell, these miRNAs will sculpt the intracellular landscape, as their activity will ultimately affect levels of target proteins. Using a high-throughput sequencing approach, we identified 19 novel small RNAs produced during the early hours of herpes simplex virus type 1 (HSV-1) infection in epithelial cells. Six of the novel RNAs had predicted folds characteristic of miRNAs. One of the six, miR-92944, which resides in the 5' UTR of the ul42 gene in the sense orientation, was confirmed as a bona fide miRNA by RT-PCR and stem-loop PCR analysis. Northern blot analysis was used to observe the precursor forms of miR-92944. Viral mutants that do not produce miR-92944 exhibited significant reductions in viral titers in both single and multi-step growth analysis and a fourfold reduction in plaque size. The miR-92944 mutants produce wild-type levels of ICP4, UL42, VP5, and gC proteins contain no additional changes in the DNA sequence surrounding the site of mutagenesis. The defective phenotype of miR-92944 mutants was complemented in V42.3 cells, which contain the 5'UTR of ul42. We also found that miR-H1 expression was diminished in cells infected with the miR-92944 mutant virus. This study provides new information on the miRNA landscape during the early stages of HSV-1 infection and reveals novel targets for antagonistic molecules that may curtail the establishment of lytic or latent virus infection.

Asunto(s)

Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , ARN Viral/metabolismo , Replicación Viral , Animales , Northern Blotting , Línea Celular , Células Epiteliales/virología , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Herpesvirus Humano 1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/genética , Mutación , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Oxidative stress gives rise to an environment that can be highly damaging to proteins, lipids, and DNA. Previous studies indicate that Herpesvirus infections cause oxidative stress in cells and in tissues. The biological consequences of virus-induced oxidative stress have not been characterized. Studies from many groups indicate that proteins which have been damaged through oxidative imbalances are either degraded by the 20S proteasome in a ubiquitin-independent fashion or form aggregates that are resistant to proteolysis. We have previously shown that herpes simplex virus type 1 (HSV-1) replication was significantly enhanced in the presence of the cellular antioxidant chaperone Hsp27, indicating a possible role for this protein in managing virus-induced oxidative stress. Here we show that oxidized proteins accumulate during infections with two distantly related herpesviruses, HSV-1 and Rhesus Rhadinovirus (RRV), a close relative of the Kaposi's sarcoma-associated herpesvirus (KSHV). The presence of oxidized proteins was not entirely unexpected as oxidative stress during herpesvirus infection has been previously documented. Unexpectedly, some oxidized proteins are removed in a proteasome-dependent fashion throughout infection and others resist degradation. Oxidized proteins that resist proteolysis become sequestered in foci within the nucleus and are not associated with virus-induced chaperone enriched domains (VICE), active centers of protein quality control, but rather coincide with Hsp27-enriched foci that were previously described by our laboratory. Experiments also indicate that the accumulation of oxidized proteins is more pronounced in cells depleted for Hsp27. We propose that Hsp27 may facilitate oxidized protein turnover at VICE domains in the nucleus during infection. Hsp27 may also buffer toxic effects of highly-carbonylated, defective proteins that resist proteolysis by promoting their aggregation in the nucleus. These roles of Hsp27 during virus infection are most likely not mutually exclusive.

Asunto(s)

Infecciones por Herpesviridae/metabolismo , Estrés Oxidativo , Proteínas/metabolismo , Infecciones Tumorales por Virus/metabolismo , Animales , Chlorocebus aethiops , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Herpesvirus Humano 1 , Humanos , Chaperonas Moleculares/metabolismo , Oxidación-Reducción , Complejo de la Endopetidasa Proteasomal/metabolismo , Carbonilación Proteica , Desnaturalización Proteica , Rhadinovirus , Células Vero

RESUMEN

Chaperone-enriched domains are formed in the nuclei of cells lytically infected with herpes simplex virus type 1 (HSV-1). These domains, called VICE, for virus induced chaperone enriched, contain Hsc70, Hsp70, Hsp40, Hsp90, polyubiquitinated proteins, and components of the proteasome machinery. Accumulating evidence indicates that these sites may be utilized during infection to sequester misfolded, modified, or otherwise unwanted proteins away from viral replication compartments, sites of robust transcription, DNA synthesis, and capsid maturation. To further explore the role of cellular chaperones and VICE domains during HSV-1 infection, we have analyzed the cytoprotective chaperone Hsp27. Here we present evidence that Hsp27, which is known to possess several antioxidant functions, is rapidly reorganized and modified at early stages in response to HSV-1 infection and signaling from the mitogen-activated protein kinase p38. Immunofluorescence analysis and fractionation experiments reveal disparate subcellular localizations of nonphosphorylated and phosphorylated forms of Hsp27 during wild-type HSV-1 infection. Unmodified forms of Hsp27 are localized in nuclear foci that are outside of replication compartments, adjacent to VICE domains, and in the cytoplasm. Conversely, we find that phosphorylated forms of Hsp27 are localized exclusively in the cytoplasm. Last, in cells depleted of all forms of Hsp27, virus replication is significantly reduced.

Asunto(s)

Proteínas de Choque Térmico HSP27/metabolismo , Herpes Simple/metabolismo , Chaperonas Moleculares/metabolismo , Replicación Viral , Animales , Núcleo Celular/química , Chlorocebus aethiops , Citoplasma/química , Herpesvirus Humano 1 , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Células Vero , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Hypoxic cancer cells are refractory to conventional chemotherapy. Herpes simplex virus type-1 (HSV-1)-derived oncolytic viruses are safe for therapy since they lack the neurovirulence gene ICP34.5. Cancer cells containing high MEK activity are permissive to the HSV-1-derived oncolytic virus, R3616. Considering that hypoxia increases MEK activity, we determined whether hypoxic MDA-MB-231 and MCF-7 cells were more permissive to R3616, compared to normoxic cells. We observed nine-fold higher (3.5 x 10e6 pfu/4 x 10e5 pfu/ml) titers in MDA-MB-231 hypoxic cells compared to normoxic; however, hypoxic MCF-7 cells did not yield higher R3616 titers. Markers for early and late viral infection were consistent with this result: (1) virus-induced chaperone-enriched (VICE) domains were observed in MDA-MB-231 cells, and (2) the HSV-1 glycoprotein C (gC), a protein produced late in infection, accumulated in hypoxic MDA-MB-231 cells. Thus, oncolytic R3616 virus may target hypoxic p53(-) breast cancer cells.

Asunto(s)

Neoplasias de la Mama/terapia , Herpesvirus Humano 1/fisiología , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Hipoxia de la Célula , Línea Celular Tumoral , ADN Viral/metabolismo , Femenino , Herpesvirus Humano 1/genética , Humanos , Virus Oncolíticos/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Replicación Viral

RESUMEN

Many viruses and bacteriophage utilize chaperone systems for DNA replication and viral morphogenesis. We have previously shown that in the herpes simplex virus type 1 (HSV-1)-infected cell nucleus, foci enriched in the Hsp70/Hsp40 chaperone machinery are formed adjacent to viral replication compartments (A. D. Burch and S. K. Weller, J. Virol. 78:7175-7185, 2004). These foci have now been named virus-induced chaperone-enriched (VICE) foci. Since the Hsp90 chaperone machinery is known to engage the Hsp70/Hsp40 system in eukaryotes, the subcellular localization of Hsp90 in HSV-1-infected cells was analyzed. Hsp90 is found within viral replication compartments as well as in the Hsp70/Hsp40-enriched foci. Geldanamycin, an inhibitor of Hsp90, results in decreased HSV-1 yields and blocks viral DNA synthesis. Furthermore, we have found that the viral DNA polymerase is mislocalized to the cytoplasm in both infected and transfected cells in the presence of geldanamycin. Additionally, in the presence of an Hsp90 inhibitor, proteasome-dependent degradation of the viral polymerase was detected by Western blot analysis. These data identify the HSV-1 polymerase as a putative client protein of the Hsp90 chaperone system. Perturbations in this association appear to result in degradation, aberrant folding, and/or intracellular localization of the viral polymerase.

Asunto(s)

Núcleo Celular/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas HSP90 de Choque Térmico/fisiología , Herpesvirus Humano 1/fisiología , Proteínas Virales/metabolismo , Animales , Benzoquinonas , Chlorocebus aethiops , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas , Quinonas/farmacología , Células Vero , Replicación Viral

RESUMEN

Herpes simplex virus type 1 (HSV-1) encodes a portal protein that forms a large oligomeric structure believed to provide the conduit for DNA entry and exit from the capsid. Chaperone proteins often facilitate the folding and multimerization of such complex structures. In this report, we show that cellular chaperone proteins, components of the 26S proteasome, and ubiquitin-conjugated proteins are sequestered in discrete foci in the nucleus of the infected cell. The immediate-early viral protein ICP0 was shown to be necessary to establish these foci at early times during infection and sufficient to redistribute chaperone molecules in transfected cells. Furthermore, we found that not only is the portal protein, UL6, localized to these sites during infection, but it is also a substrate for ubiquitin modification. Our results suggest that HSV-1 has evolved an elegant mechanism for facilitating protein quality control at specialized foci within the nucleus.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Herpesvirus Humano 1/patogenicidad , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Ubiquitina/metabolismo , Animales , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas del Choque Térmico HSC70 , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Ubiquitina-Proteína Ligasas , Células Vero , Proteínas Virales

RESUMEN

Putative conformational switching and inhibitory regions in the Microviridae external scaffolding protein were investigated. Substitutions for glycine 61, hypothesized to promote a postdimerization conformational switch, have dominant lethal phenotypes. In previous studies, chimeric alpha3/phiX174 proteins for structures alpha-helix 1 and loop 6/alpha-helix 7 inhibited phiX174 morphogenesis when expressed from high copy number plasmids. To determine if inhibition was due to overexpression, chimeric genes were constructed into the phiX174 genome. In coinfections with wild-type, protein ratios would be 1:1. The helix 1 chimera has a recessive lethal phenotype; thus, overexpression confers inhibition. In single infections, the mutant cannot form procapsids, suggesting that helix 1 mediates the initial recognition of structural proteins. The lethal chimeric helix 7 protein has a dominant phenotype. Alone, the mutant forms defective procapsids, suggesting a later morphogenetic defect. The results of second-site genetic analyses indicate that the capsid-external scaffolding protein interface is larger than revealed in the crystal structure.

Asunto(s)

Proteínas de la Cápside/química , Microviridae/química , Ensamble de Virus , Secuencia de Aminoácidos , Bacteriófago phi X 174/química , Proteínas de la Cápside/genética , Codón , Dimerización , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes de Fusión/química