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1.
J Gen Virol ; 91(Pt 2): 470-82, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19846675

RESUMO

Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2alpha phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-DeltaC7L) lacked late gene expression, which could be rescued by the function of host-range factor K1 or C7. It was demonstrated that viral gene expression was blocked after viral DNA replication and that it was independent of apoptosis induction. Furthermore, it was found that eIF2alpha phosphorylation in MVA-DeltaC7L-infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts lacking PKR function, and it was shown that this was not due to reduced E3L gene expression. The block of eIF2alpha phosphorylation by C7 could be complemented by K1 in cells infected with MVA-DeltaC7L encoding a reinserted K1L gene (MVA-DeltaC7L-K1L). Importantly, these data illustrated that eIF2alpha phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and probably also for VACV replication in a diverse set of cell types.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Viral da Expressão Gênica , Vaccinia virus/metabolismo , Vacínia/metabolismo , Proteínas Virais/metabolismo , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Cricetinae , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Camundongos , Fosforilação , Vacínia/enzimologia , Vacínia/genética , Vacínia/virologia , Vaccinia virus/genética , Proteínas Virais/genética , eIF-2 Quinase/genética
2.
J Virol ; 83(12): 6029-38, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19357172

RESUMO

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Delta68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Delta68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Delta68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein.


Assuntos
Anquirinas/genética , Replicação do DNA , Vaccinia virus/fisiologia , Proteínas Virais/genética , Replicação Viral , Animais , Motivos F-Box , Deleção de Genes , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Células NIH 3T3 , Fases de Leitura Aberta , Fenótipo , RNA Viral/biossíntese , Deleção de Sequência , Transcrição Gênica , Vaccinia virus/genética , Vaccinia virus/crescimento & desenvolvimento
3.
Virology ; 374(2): 234-9, 2008 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-18353424

RESUMO

The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF complex formation might be important for the viral life cycle.


Assuntos
Anquirinas , Sequência Conservada , Orthopoxvirus/metabolismo , Proteínas Ligases SKP Culina F-Box , Sequência de Aminoácidos , Animais , Anquirinas/química , Anquirinas/genética , Anquirinas/metabolismo , Motivos F-Box/genética , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Orthopoxvirus/genética , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Vaccinia virus/genética , Vaccinia virus/metabolismo
4.
AIDS Res Hum Retroviruses ; 24(12): 1527-36, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19018669

RESUMO

HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.


Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/isolamento & purificação , HIV-1/fisiologia , Receptores CCR5/metabolismo , África Austral , Substituição de Aminoácidos/genética , Antígenos CD4/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica
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