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1.
Proc Natl Acad Sci U S A ; 118(30)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34282019

RESUMO

N 6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

2.
Viruses ; 13(3)2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671095

RESUMO

The APOBEC family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes - APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new host-pathogen conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.


Assuntos
Desaminases APOBEC/genética , Herpesviridae/genética , Animais , Replicação do DNA/genética , Vírus de DNA/genética , DNA Viral/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Replicação Viral/genética
3.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31534038

RESUMO

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.


Assuntos
Citidina Desaminase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Citosina Desaminase/metabolismo , Células HEK293 , Herpes Simples , Infecções por Herpesviridae , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas/metabolismo , Replicação Viral
4.
EMBO J ; 35(13): 1385-99, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27234299

RESUMO

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.


Assuntos
Herpesvirus Humano 1/patogenicidade , Proteínas Imediatamente Precoces/metabolismo , Evasão da Resposta Imune , Interferon Tipo I/antagonistas & inibidores , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Mapeamento de Interação de Proteínas
5.
J Virol ; 89(5): 2892-905, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25540385

RESUMO

UNLABELLED: Numerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27 dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture medium. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore-unappreciated role in modulating the efficiency of progeny virion release. IMPORTANCE: ICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life cycle. Although ICP27 homologs are encoded by all known members of the Herpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release.


Assuntos
Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Virais/metabolismo , Vírion/fisiologia , Liberação de Vírus , Animais , Linhagem Celular , Humanos , Ensaio de Placa Viral , Replicação Viral
6.
J Virol ; 87(16): 8940-51, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23740995

RESUMO

Leptomycin B (LMB) is a highly specific inhibitor of CRM1, a cellular karyopherin-ß that transports nuclear export signal-containing proteins from the nucleus to the cytoplasm. Previous work has shown that LMB blocks herpes simplex virus 1 (HSV-1) replication in Vero cells and that certain mutations in viral immediate early protein ICP27 can confer LMB resistance. However, little is known of the molecular mechanisms involved. Here we report that HSV-2, a close relative of HSV-1, is naturally resistant to LMB. To see whether the ICP27 gene determines this phenotypic difference, we generated an HSV-1 mutant that expresses the HSV-2 ICP27 instead of the HSV-1 protein. This recombinant was fully sensitive to LMB, indicating that one or more other viral genes must be important in determining HSV-2's LMB-resistant phenotype. In additional work, we report several findings that shed light on how HSV-1 ICP27 mutations can confer LMB resistance. First, we show that LMB treatment of HSV-1-infected cells leads to suppression of late viral protein synthesis and a block to progeny virion release. Second, we identify a novel type of ICP27 mutation that can confer LMB resistance, that being the addition of a 100-residue amino-terminal affinity purification tag. Third, by studying infections where both LMB-sensitive and LMB-resistant forms of ICP27 are present, we show that HSV-1's sensitivity to LMB is dominant to its resistance. Together, our results suggest a model in which the N-terminal portion of ICP27 mediates a nonessential activity that interferes with HSV-1 replication when CRM1 is inactive. We suggest that LMB resistance mutations weaken or abrogate this activity.


Assuntos
Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Farmacorresistência Viral , Ácidos Graxos Insaturados/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Recombinação Genética , Replicação Viral/efeitos dos fármacos
7.
Future Microbiol ; 8(3): 311-21, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23464370

RESUMO

During its productive infection, HSV-1 dramatically remodels the architecture and physiology of the host cell nucleus. The immediate-early proteins, the first viral proteins to be expressed during infection, are key players in this process. Here, we review the known properties and functions of immediate-early protein ICP22. Although this polypeptide has received less attention than other immediate-early proteins, the published evidence indicates that it mediates several striking changes to important host nuclear systems, including those involved in RNA polymerase II transcription, cell cycle regulation and protein quality control. Recent genetic analyses suggest that these alterations can promote HSV-1 productive infection. Thus, future work on ICP22 is likely to reveal novel mechanisms by which herpesviruses, and possibly other DNA viruses, manipulate the host cell nucleus to enhance their replication.


Assuntos
Núcleo Celular/virologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/metabolismo , Replicação Viral , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos
8.
PLoS One ; 6(7): e22638, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21818356

RESUMO

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone.


Assuntos
Colo do Útero/patologia , Colo do Útero/virologia , Epitélio/patologia , Epitélio/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Simplexvirus/fisiologia , Antígenos Virais/imunologia , Agregação Celular , Células Cultivadas , Suscetibilidade a Doenças , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Fibroblastos/patologia , Fibroblastos/virologia , Células Gigantes/patologia , Células Gigantes/virologia , Infecções por HIV/complicações , Infecções por HIV/patologia , HIV-1/imunologia , Humanos , Imuno-Histoquímica , Inflamação/complicações , Inflamação/patologia , Membrana Mucosa/patologia , Membrana Mucosa/virologia , Técnicas de Cultura de Órgãos , Pré-Menopausa , Simplexvirus/imunologia
9.
J Virol ; 85(11): 5685-90, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21411532

RESUMO

ICP27 is an essential herpes simplex virus 1 (HSV-1) regulatory protein that enhances viral gene expression. Although it is predominantly nuclear, it shuttles to the cytoplasm during infection using an N-terminal nuclear export signal (NES). We previously engineered an NES-negative ICP27 mutant, dLeu, that replicates poorly in cultured cells. In this study, we isolated dLeuR, a growth-competent revertant of dLeu. We show that dLeuR possesses one or more extragenic mutations that enhance ICP27 transcription, leading to overexpression of the mutant protein and restoration of viral growth. This work provides evidence of a novel pathway regulating transcription of the ICP27 gene.


Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Mutação de Sentido Incorreto , Supressão Genética , Transcrição Genética , Replicação Viral , Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento
10.
J Virol ; 84(6): 2707-18, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20042503

RESUMO

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the cis-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a cis-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp "silencing element" (SE) in the 5' portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.


Assuntos
Sequência de Bases , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Chlorocebus aethiops , Replicação do DNA , Inativação Gênica , Herpes Simples/genética , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Células Vero , Proteínas do Envelope Viral/genética
11.
J Virol ; 84(5): 2384-94, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20032172

RESUMO

During productive infection, herpes simplex virus type 1 (HSV-1) induces the formation of discrete nuclear foci containing cellular chaperone proteins, proteasomal components, and ubiquitinated proteins. These structures are known as VICE domains and are hypothesized to play an important role in protein turnover and nuclear remodeling in HSV-1-infected cells. Here we show that VICE domain formation in Vero and other cells requires the HSV-1 immediate-early protein ICP22. Since ICP22 null mutants replicate efficiently in Vero cells despite being unable to induce VICE domain formation, it can be concluded that VICE domain formation is not essential for HSV-1 productive infection. However, our findings do not exclude the possibility that VICE domain formation is required for viral replication in cells that are nonpermissive for ICP22 mutants. Our studies also show that ICP22 itself localizes to VICE domains, suggesting that it could play a role in forming these structures. Consistent with this, we found that ICP22 expression in transfected cells is sufficient to reorganize the VICE domain component Hsc70 into nuclear inclusion bodies that resemble VICE domains. An N-terminal segment of ICP22, corresponding to residues 1 to 146, is critical for VICE domain formation in infected cells and Hsc70 reorganization in transfected cells. We previously found that this portion of the protein is dispensable for ICP22's effects on RNA polymerase II phosphorylation. Thus, ICP22 mediates two distinct regulatory activities that both modify important components of the host cell nucleus.


Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Animais , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Chlorocebus aethiops , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/genética , Corpos de Inclusão Intranuclear/química , Corpos de Inclusão Intranuclear/metabolismo , Fosforilação , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Células Vero
12.
J Virol ; 83(1): 128-39, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18971282

RESUMO

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , RNA Polimerase II/metabolismo , Animais , Linhagem Celular , Núcleo Celular/química , Chlorocebus aethiops , Herpesvirus Humano 1/genética , Humanos , Microscopia de Fluorescência , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Sinais de Localização Nuclear
13.
J Virol ; 83(4): 1767-77, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19073744

RESUMO

The herpes simplex virus type 1 (HSV-1) protein ICP27 has been implicated in a variety of functions important for viral replication including host shutoff, viral gene expression, activation of mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK), and apoptosis inhibition. In the present study we sought to examine the functions of ICP27 in the absence of viral infection by creating stable HeLa cell lines that inducibly express ICP27. Here, we characterize two such cell lines and show that ICP27 expression is associated with a cellular growth defect. The observed defect is caused at least in part by the induction of apoptosis as indicated by caspase-3 activation, annexin V staining, and characteristic changes in cellular morphology. In an effort to identify the function of ICP27 responsible for inducing apoptosis, we show that ICP27 expression is sufficient to activate p38 signaling to a level that is similar to that observed during wild-type HSV-1 infection. However, ICP27 expression alone is unable to lead to a strong activation of JNK signaling. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling is responsible for the observed induction of apoptosis in the induced cell lines. Our findings suggest that during viral infection, ICP27 activates p38 and JNK signaling pathways via two distinct mechanisms. ICP27 directly activates p38 signaling, leading to stimulation of the host cell apoptotic pathways. In contrast, robust activation of JNK signaling by ICP27 requires one or more delayed early or late viral gene products and may be associated with the inhibition of apoptosis.


Assuntos
Apoptose , Herpesvirus Humano 1/patogenicidade , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anexina A5/metabolismo , Caspase 3/metabolismo , Células HeLa , Humanos
14.
J Virol ; 82(15): 7443-55, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18495765

RESUMO

We previously showed that herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP27 can posttranscriptionally stimulate mRNA accumulation from a transfected viral late gene encoding glycoprotein C (gC) (K. D. Perkins, J. Gregonis, S. Borge, and S. A. Rice, J. Virol. 77:9872-9884, 2003). We began this study by asking whether ICP27 homologs from other herpesviruses can also mediate this activity. Although the homologs from varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) were inactive, the homolog from bovine herpesvirus 4 (BHV-4), termed HORF1/2, was a very efficient transactivator. Surprisingly, most of the mRNA produced via HORF1/2 transactivation was 225 nucleotides shorter than expected due to the removal of a previously undescribed intron from the gC transcript. We found that the gC mRNA produced in the absence of transactivation was also mostly spliced. In contrast, gC mRNA produced by ICP27 transactivation was predominantly unspliced. Based on these results, we conclude that ICP27 has two distinct effects on the transfected gC gene: it (i) stimulates mRNA accumulation and (ii) promotes the retention of an intron. Interestingly, the spliced transcript encodes a variant of gC that lacks its transmembrane domain and is secreted from transfected cells. As the gC splicing signals are conserved among several HSV-1 strains, we investigated whether the variant gC is expressed during viral infection. We report here that both the spliced transcript and its encoded protein are readily detected in Vero cells infected with three different laboratory strains of wild-type HSV-1. Moreover, the variant gC is efficiently secreted from infected cells. We have designated this alternate form of the protein as gCsec. As the extracellular domain of gC is known to bind heparan sulfate-containing proteoglycans and to inhibit the complement cascade via an interaction with complement component C3b, we speculate that gCsec could function as a secreted virulence factor.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas do Envelope Viral/biossíntese , Animais , Linhagem Celular , Chlorocebus aethiops , Herpesvirus Bovino 4 , Humanos , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Transativadores/metabolismo , Proteínas não Estruturais Virais/metabolismo
15.
J Virol ; 82(1): 268-77, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17959681

RESUMO

Early in infection, herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 localize to the nucleus, where they stimulate viral transcription. Later in infection, ICP0 and to a lesser extent ICP4 accumulate in the cytoplasm, but their biological role there is unknown. Previously, it was shown that the cytoplasmic localization of ICP0/4 requires the multifunctional IE protein ICP27, which is itself an activator of viral gene expression. Here, we identify a viral ICP27 mutant, d3-4, which is unable to efficiently localize ICP0 and ICP4 to the cytoplasm but which otherwise resembles wild-type HSV-1 in its growth and viral gene expression phenotypes. These results genetically separate the function of ICP27 that affects ICP0/4 localization from its other functions, which affect viral growth and gene expression. As both ICP0 and ICP4 are known to be minor virion components, we used d3-4 to test the hypothesis that the cytoplasmic localization of these proteins is required for their incorporation into viral particles. Consistent with this conjecture, d3-4 virions were found to lack ICP0 in their tegument and to have greatly reduced levels of ICP4. Thus, the cytoplasmic localization of ICP0 and ICP4 appears to be a prerequisite for the assembly of these important transcriptional regulatory proteins into viral particles. Furthermore, our results show that ICP27 plays a previously unrecognized role in determining the composition of HSV-1 virions.


Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Vírion/química , Montagem de Vírus/fisiologia , Citoplasma/química , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/genética , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Montagem de Vírus/genética , Replicação Viral/genética , Replicação Viral/fisiologia
16.
J Virol ; 81(10): 5091-101, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17344289

RESUMO

During eukaryotic mRNA transcription, the synthetic activity and mRNA processing factor interactions of RNA polymerase II (RNAP II) are regulated by phosphorylation of its carboxyl-terminal domain (CTD), with modification occurring primarily on serines 2 and 5 of the CTD. We previously showed that herpes simplex virus type 1 (HSV-1) infection rapidly triggers the loss of RNAP II forms bearing serine 2 phosphorylation (Ser-2P RNAP II). Here we show that the HSV-1 immediate-early (IE) protein ICP22 is responsible for this effect during the IE phase of infection. This activity does not require the viral UL13 protein kinase, which is required for several other regulatory functions of ICP22. Additionally, we show that transient expression of ICP22 can trigger the loss of Ser-2P RNAP II in transfected cells. Thus, the ability of ICP22 to cause the loss of Ser-2 RNAP II does not require other viral factors or the context of the infected cell. Expression of the HSV-1 ICP22-related protein US1.5, which corresponds to residues 147 to 420 of ICP22, also triggers a loss of Ser-2P RNAP II in transfected cells, whereas expression of the varicella-zoster virus ICP22 homolog, ORF63, does not. Our study also provides evidence for a second, viral late gene-dependent pathway that triggers loss of Ser-2P RNAP II in infected cells, consistent with the recent work of Dai-Ju et al. (J. Q. Dai-Ju, L. Li, L. A. Johnson, and R. M. Sandri-Goldin, J. Virol. 80:3567-3581, 2006). Therefore, it appears that HSV-1 has evolved redundant mechanisms for triggering the loss of a specific phosphorylated form of RNAP II.


Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/fisiologia , RNA Polimerase II/metabolismo , Animais , Chlorocebus aethiops , Immunoblotting , Microscopia de Fluorescência , Fosforilação , Proteínas Quinases/fisiologia , Serina/metabolismo , Células Vero , Proteínas do Envelope Viral/fisiologia , Proteínas Virais Reguladoras e Acessórias
17.
J Virol ; 80(19): 9381-90, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16973544

RESUMO

The immediate-early regulatory protein ICP22 is required for efficient replication of herpes simplex virus type 1 in some cell types (permissive) but not in others (restrictive). In mice infected via the ocular route, the pathogenesis of an ICP22- virus, 22/n199, was altered relative to that of wild-type virus. Specifically, tear film titers of 22/n199-infected mice were significantly reduced at 3 h postinfection relative to those of mice infected with wild-type virus. Further, 22/n199 virus titers were below the level of detection in trigeminal ganglia (TG) during the first 9 days postinfection. On day 30 postinfection, TG from 22/n199-infected mice contained reduced viral genome loads and exhibited reduced expression of latency-associated transcripts and reduced reactivation efficiency relative to TG from wild-type virus-infected mice. Notably, the first detectable alteration in the pathogenesis of 22/n199 in these tests occurred in the eye prior to the onset of nascent virus production. Thus, ICP22- virions appeared to be degraded, cleared, or adsorbed more rapidly than wild-type virions, implying potential differences in the composition of the two virion types. Analysis of the protein composition of purified extracellular virions indicated that ICP22 is not a virion component and that 22/n199 virions sediment at a reduced density relative to wild-type virions. Although similar to wild-type virions morphologically, 22/n199 virions contain reduced amounts of two gamma2 late proteins, US11 and gC, and increased amounts of two immediate-early proteins, ICP0 and ICP4, as well as protein species not detected in wild-type virions. Although ICP22- viruses replicate to near-wild-type levels in permissive cells, the virions produced in these cells are biochemically and physically different from wild-type virions. These virion-specific differences in ICP22- viruses add a new level of complexity to the functional analysis of this immediate-early viral regulatory protein.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/química , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Vírion/química , Vírion/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral/genética , Herpes Simples/genética , Herpes Simples/patologia , Humanos , Proteínas Imediatamente Precoces/isolamento & purificação , Cinética , Masculino , Camundongos , Mutação/genética , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia , Proteínas Virais Reguladoras e Acessórias , Vírion/genética , Vírion/isolamento & purificação
18.
Virology ; 352(2): 368-79, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16780914

RESUMO

It was previously shown that herpes simplex virus type 1 (HSV-1) is sensitive to leptomycin B (LMB), an inhibitor of nuclear export factor CRM1, and that a single methionine to threonine change at residue 50 (M50T) of viral immediate-early (IE) protein ICP27 can confer LMB resistance. In this work, we show that deletion of residues 21-63 from ICP27 can also confer LMB resistance. We further show that neither the M50T mutation nor the presence of LMB affects the nuclear shuttling activity of ICP27, suggesting that another function of ICP27 determines LMB resistance. A possible clue to this function emerged when it was discovered that LMB treatment of HSV-1-infected cells dramatically enhances the cytoplasmic accumulation of two other IE proteins, ICP0 and ICP4. This effect is completely dependent on ICP27 and is reversed in cells infected with LMB-resistant mutants. Moreover, LMB-resistant mutations in ICP27 enhance the nuclear localization of ICP0 and ICP4 even in the absence of LMB, and this effect can be discerned in transfected cells. Thus, the same amino (N)-terminal region of ICP27 that determines sensitivity to LMB also enhances ICP27's previously documented ability to promote the cytoplasmic accumulation of ICP4 and ICP0. We speculate that ICP27's effects on ICP4 and ICP0 may contribute to HSV-1 LMB sensitivity.


Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Citoplasma/virologia , Farmacorresistência Viral , Ácidos Graxos Insaturados/farmacologia , Genes Virais , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Mutação Puntual , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Células Vero
19.
J Virol ; 79(17): 11323-34, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16103184

RESUMO

Previous studies have shown that herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP II), creating a new form of the enzyme known as RNAP II(I). However, the specific phosphorylation changes induced by HSV-1 have not been characterized. In this study, we used phospho-specific anti-CTD antibodies to probe the structure of the postinfection RNAP II. We find that RNAP II(I) is phosphorylated on serine-5 (Ser-5) of the CTD consensus repeat but generally lacks phosphorylation on serine-2 (Ser-2). Since Ser-2 phosphorylation is normally associated with efficient transcriptional elongation and the recruitment of pre-mRNA processing factors, our results suggest that RNAP II(I) may have altered elongation properties and decreased interactions with the mRNA processing machinery. The viral factors responsible for the reduction in Ser-2 CTD phosphorylation were studied. We found that viral immediate-early (IE) gene expression is required and sufficient, in the context of infection, for loss of Ser-2 phosphorylation. However, studies with viral mutants failed to implicate a single IE protein (among ICP0, ICP4, ICP22, and ICP27) in this process. Although most Ser-2-phosphorylated RNAP II is lost after infection, our immunofluorescence analyses identified a small subfraction that escapes loss and relocalizes to splicing antigen-rich nuclear speckles. A similar phenomenon is seen in uninfected cells after various treatments that inhibit RNAP II transcription. We hypothesize that the HSV-1-induced relocalization of residual Ser-2-phosphorylated RNAP II to nuclear speckles reflects a host response to the inhibition of cellular gene transcription.


Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , RNA Polimerase II/metabolismo , Animais , Linhagem Celular , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Fosforilação , Estrutura Terciária de Proteína , Serina , Transcrição Genética , Replicação Viral
20.
J Virol ; 77(18): 9872-84, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12941897

RESUMO

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that stimulates viral mRNA expression from many viral delayed-early and late genes during infection. One HSV-1 late gene which is highly dependent on ICP27 during infection is that encoding the glycoprotein C (gC). Here we report that the gC gene is specifically transactivated by ICP27 in transfected Vero cells. Using various gC plasmid constructs, we show that ICP27's stimulatory effects are independent of the gC gene's endogenous promoter and polyadenylation site. This suggests that ICP27-responsive elements lie in the transcribed body of the gC gene. We also show that transactivation of the gC gene by ICP27 is independent of other viral proteins, as ICP27 alone can transactivate the gC gene when its transcription is mediated by the human cytomegalovirus immediate-early gene promoter. However, when gC gene expression is driven by its endogenous promoter, the stimulatory effect of ICP27 requires additional transactivators. To explore the level at which ICP27 transactivates the gC gene, we established stably transfected Vero cell lines that have integrated copies of the gC gene under control of the cytomegalovirus immediate-early gene promoter. These gC genes are not constitutively expressed but can be efficiently induced by HSV-1 infection. Using nuclear run-on transcription assays, we show that transcriptional induction of the stably transfected genes is ICP27 independent. In contrast, accumulation of gC mRNA is very highly dependent on ICP27. Together, these results demonstrate that ICP27 posttranscriptionally activates mRNA expression from a biologically relevant viral target gene.


Assuntos
Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/fisiologia , Poliadenilação , Regiões Promotoras Genéticas , Ativação Transcricional , Proteínas do Envelope Viral/genética , Animais , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Transfecção , Ubiquitina-Proteína Ligases , Células Vero
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