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PubMed1995

Separation of sequence requirements for HSV-1 Vmw110 multimerisation and interaction with a 135-kDa cellular protein.

Herpes simplex virus type 1 immediate-early polypeptide Vmw110 (ICP0) is a general transactivator of gene expression in transfection assays and is required for the fully efficient onset of viral lytic replication. It has also been implicated in the process of viral reactivation from latency. Its mechanism of action is unknown, but any involvement in latency requires interactions between viral and host factors. We have previously shown that Vmw110 binds to a 135-kDa cellular protein. In this paper we define a short region towards the C-terminal end of Vmw110 that is required for the 135-kDa protein interaction in virus-infected cells and in vitro. We also confirm that the C-terminal region of Vmw110 contains residues that are responsible for the multimerisation of the protein; these sequences are at least partially distinct from those involved in 135-kDa binding. Both multimerisation and 135-kDa protein interaction are required for full viral infectivity, and elimination of these functions affects the normal interactions between Vmw110 and cellular nuclear structures that contain the PML protein.

Animals↗
PubMed1998

Rescue of the Epstein-Barr virus BZLF1 mutant, Z(S186A), early gene activation defect by the BRLF1 gene product.

Expression of the Epstein-Barr virus (EBV) immediate-early protein, BZLF1 (Z), is sufficient to disrupt viral latency. Z transcriptionally activates the EBV early genes by binding to upstream Z-responsive elements (ZREs). Recently, a serine-to-alanine mutation of Z residue 186 (within the basic DNA binding domain) was shown to inhibit the ability of Z to induce lytic infection in latently infected cells, although the Z(S186A) mutant could still bind several known ZREs and activated an early EBV promoter (BMRF1) in transient reporter gene assays (Francis, A. L., Gradoville, L., and Miller, G. (1997). J. Virol. 71, 3054-3061). We now show that a specific deficiency in the ability to bind to ZRE elements in the immediate-early BRLF1 promoter may account for the inability of Z(S186A) to activate BRLF1 expression. Furthermore, we demonstrate that the ability of Z(S186A) to induce early BMRF1 and BHRF1 gene expression is rescued by cotransfection with a BRLF1 expression vector. However, the Z(S186A)/BRLF1 (R) combination cannot induce full lytic replication, suggesting that Z(S186A) may also be deficient in a replication-specific function. These results suggest that in the context of the intact viral genome, both Z and R expression are required for activation of early gene transcription in latently infected cells.

Antigens, Viral↗
PubMed2000

Attachment of coxsackievirus B3 variants to various cell lines: mapping of phenotypic differences to capsid protein VP1.

The coxsackievirus B3 (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property, the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VP1 capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and I92 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VP1 are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 PD and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces.

Amino Acid Substitution↗
PubMed1989

Size and stability of the Epstein-Barr virus major internal repeat (IR-1) in Burkitt's lymphoma and lymphoblastoid cell lines.

We have used field inversion gel electrophoresis to survey EBV strains for the size of the major internal repeat, IR-1, and estimate the number of 3.1-kb repeat units present. The B95-8 strain of EBV was estimated to contain 8.6 repeats. The repeat number varies considerably among naturally occurring isolates around a mean of six repeats. Some cell lines harbored multiple viral genomes with differing numbers of repeats and our results suggest that the repeat number in IR-1 is more likely to change during lytic replication than during latency. The Jijoye strain had 6.6 repeats and the Jijoye deletion mutant clone P3HR-1 retained 5.9 repeats setting the size of the P3HR-1 deletion at 6.8 kb. Thus, the nonimmortalizing mutant has retained all of the W1 and W2 exons of the immortalizing parent and has lost only the 3' unique exons of EBNA4 and all of EBNA2.

Antigens, Viral↗
PubMed1988

A novel EBNA-1 titration method and putative anti-EBNA-1 protein.

A novel and rapid EBNA-1 titration method has been developed which uses immunoprecipitation of specific DNA-protein complexes with EBNA-1-positive serum. The method is more sensitive than the conventional immunofluorescence method and has potential value as a diagnostic reagent for clinical laboratories. TPA induction of putative anti-EBNA-1 protein of cellular origin is discussed, which may play a key role for the shift from latent to lytic replication of EBV.

Antibodies, Viral↗
PubMed1988

Susceptibility to parvovirus Minute virus of mice as a function of the degree of host cell transformation: little effect of simian virus 40 infection and phorbol ester treatment.

Transformation of mouse fibroblasts by simian virus 40 (SV40) or certain oncogenes enhances their susceptibility to the lytic replication of Minute virus of mice (MVM), a non-defective parvovirus. It was investigated whether this cytotoxic action of MVM can also be potentiated by two types of incomplete and reversible cell transformation induced either by SV40 infection or by exposure to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Although the latter two treatments trigger the expression of several traits of the transformed phenotype, they do not significantly enhance cell permissiveness to MVM.

Animals↗
PubMed1994

Tissue tropism and species specificity of poliovirus infection.

Paralytic poliomyelitis is caused by the destruction of neurons in the central nervous system during the lytic replication of poliovirus. The human gene for the poliovirus receptor has been cloned recently, and transgenic mice generated that are susceptible to this virus. This has allowed the critical amino acids that determine the specificity of poliovirus to be identified.

Amino Acid Sequence↗
PubMed2005

Spindle cells and their role in Kaposi's sarcoma.

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.

Cell Lineage↗
PubMed2004

BKV and SV40 infection of human kidney tubular epithelial cells in vitro.

The interaction of BKV with its natural target cells, human kidney epithelial cells, has not been studied. In vitro infections of human primary kidney epithelial cells were performed to investigate a BKV infection in its natural host cell. BKV undergoes a lytic replication cycle in this system: high levels of T antigen expression were first detected at 36 h postinfection, while viral DNA replication, capsid protein expression, and progeny virus were observed at 48 h postinfection. It was observed that the related polyomavirus SV40 is incapable of infecting human kidney epithelium except in the presence of the GM1 ganglioside, recently reported to be an SV40 receptor.

Animals↗
PubMed2006

Complete nucleotide sequence and genome analysis of bacteriophage BFK20--a lytic phage of the industrial producer Brevibacterium flavum.

The entire double-stranded DNA genome of bacteriophage BFK20, a lytic phage of the Brevibacterium flavum CCM 251--industrial producer of L-lysine--was sequenced and analyzed. It consists of 42,968 base pairs with an overall molar G + C content of 56.2%. Fifty-five potential open reading frames were identified and annotated using various bioinformatics tools. Clusters of functionally related putative genes were defined (structural, lytic, replication and regulatory). To verify the annotation of structural proteins, they were resolved by 2D gel electrophoresis and were submitted to N-terminal amino acid sequencing. Structural proteins identified included the portal and major and minor tail proteins. Based on the overall genome sequence comparison, similarities with other known bacteriophage genomes include primarily bacteriophages from Mycobacterium spp. and some regions of Corynebacterium spp. genomes--possible prophages. Our results support the theory that phage genomes are mosaics with respect to each other.

Bacteriophages↗
PubMed2005

Cellular uptake of the EBV transcription factor EB1/Zta.

A number of viral proteins have the property to penetrate into the cells when present in the extra-cellular compartment. Here, we report that the Epstein-Barr virus (EBV) transcriptional activator EB1/Zta, which is responsible for the activation of the EBV lytic replication, binds to lymphoid cells surface, is efficiently translocated and accumulates in the nucleus. The internalization of EB1/Zta is energy-dependent and shares common features with endocytosis. As the EB1/Zta was not degraded in the cells and reached the nucleus, the potential effect of its internalisation on viral reactivation was assessed.

B-Lymphocytes↗
PubMed2003

Lytic switch protein (ORF50) response element in the Kaposi's sarcoma-associated herpesvirus K8 promoter is located within but does not require a palindromic structure.

Kaposi's sarcoma-associated virus (KSHV) ORF50 protein induces lytic replication and activates the K8 promoter. We show that ORF50-induced and tetradecanoyl phorbol acetate (TPA) induced K8 transcripts initiated from the same start site. A newly identified palindrome (PAL2), containing a 12-bp response region required for ORF50-induced activation in lymphoid cells, was identified in the K8 promoter. Specific DNA binding of bacterially expressed ORF50 was not seen with the K8 promoter despite specific binding to the PAN promoter. The new palindrome shared homology with a previously described ORF50 response element (50RE(K8) and 50RE(57)). We demonstrate that the new 50RE(K8) (50RE(K8-PAL2)) is not the palindrome per se. Instead, the response element is buried within the right arm of the palindrome. We propose that the complexity of the K8 response elements reflects the complexity of mechanisms used by ORF50 during viral reactivation.

Animals↗
PubMed1998

Purification of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) and analyses of the structural proteins.

The Kaposis's sarcoma-associated herpesvirus (KSHV) infected BCBL-1 cell line, adapted to and grown in medium containing 10% horse serum, was induced to lytic replication with 12-O-tetradecanoylphorbol-13-acetate (TPA) for virus production. Supernatants from induced cells were filtered through a 0.45-microm filter and virions were concentrated by polyethylene glycol extraction and high speed centrifugation. The virus was purified by a glycerol gradient zonal centrifugation step followed by isopycnic separation using positive density negative viscosity gradients. Two visible bands were detected after the final centrifugation step: an upper band that contained a homogenous population of purified virions and a lower band that contained aggregates of purified virus and other cellular debris. Fractionation of purified virion preparations by SDS-PAGE revealed 32 bands with estimated molecular weights between 19 and 280 K in silver stained gels. The glycoprotein bands in purified virus were identified with biotinylated lectins and horseradish peroxidase-labeled streptavidin. Two lectins were used to identify the KSHV glycoproteins: concanavalin A and Ricinus communis agglutinin I. Eight distinct glycoproteins were detected with these lectins. In addition, antisera from KS patients were used to detect immunoreactive proteins in purified virions. An apparent immunodominant band of Mr 94,000 (94 K) was recognized by patients' antisera. Other proteins detected with some of the KS antisera tested corresponded to molecular weights of 57 K, 70 K, 180 K, 200 K and 240 K. The 94 K band was identified as gp94 by Endo F digestion.

Cell Line↗
PubMed2002

Activation of human herpesvirus 8 open reading frame K5 independent of ORF50 expression.

Open reading frame (ORF) 50 of human herpesvirus 8 (HHV8, Kaposi's sarcoma-associated herpesvirus) is one of the immediate-early gene and a homologue of BRLF1 gene of Epstein-Barr virus. It encodes a key switch protein to trigger viral lytic replication from latency. We have established several hybridoma clones producing monoclonal antibodies (MAbs) to the products of HHV8 ORFs. Using these antibodies, we analyzed antigen expression in a HHV8 infected cell line after treatment with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). A MAb reacted to 110 kilodalton (kDa) and 62 kDa proteins encoded by ORF50 (ORF50 protein). Kinetic studies of antigen expression by Western blotting revealed that ORF50 protein was induced as early as 6 h after TPA treatment. The proteins encoded by ORFK3, ORFK5, ORFK9, ORF59 and ORFK8.1 were not detected earlier than ORF50 protein. However, when antigen positive cells were counted by immunofluorescent antibody (IFA) test, number of ORFK5 protein positive cells were higher than that of ORF50 protein positive cells at all time after TPA or mock treatment. To confirm the results of IFA test, individual cell was analyzed by reverse transcription polymerase chain reaction. Some cells expressed ORFK5 transcript but not ORF50 transcript. Therefore, we concluded that, although ORF50 protein is a key switch protein of ORFK3, ORFK9, ORF59 and ORFK8.1 expression, it is not essential to trigger ORFK5 gene.

Antibodies, Monoclonal↗
PubMed1998

Herpes simplex virus latency and the immune response.

Following infection, herpes simplex virus establishes latency in the nervous system and recurrences of lytic replication occur periodically. Molecular events which may determine how virus enters latency, how it is maintained and what occurs during reactivation have been investigated. The role of the immune response in limiting infection of the nervous system, influencing the latent state and removing virus from peripheral sites following reactivation has also been studied.

Herpes Simplex↗
PubMed2000

Presence of human herpesvirus-8 DNA sequences and overexpression of human IL-6 and cyclin D1 in inflammatory myofibroblastic tumor (inflammatory pseudotumor).

Inflammatory myofibroblastic tumor (IMT) is composed of myofibroblasts, plasma cells, and lymphocytes. Cytokines are possibly involved in its pathogenesis. Human herpesvirus-8 (HHV-8) encodes cell cycle regulatory and signaling proteins. A combination of nested PCR with several negative controls and Southern blot methods showed the presence of HHV-8 DNA in seven cases of IMT. Additionally, strong expression was demonstrated by in situ hybridization in many tumoral nuclei. Most of the myofibroblasts in all of the cases were immunoreactive for human IL-6 and cyclin D1. These cytokines probably have a paracrine action and may sustain myofibroblastic growth. HHV-8 could play an essential role in triggering IMT development by a local reactivation of viral lytic replication. The relationship between HHV-8 and immunosuppression status as the only associated cause for tumorigenesis should be revised.

Adult↗
PubMed2005

Induced structure of a helical switch as a mechanism to regulate enzymatic activity.

Herpesviruses encode a protease that is activated by homodimerization at high enzyme concentrations during lytic replication. The homodimer contains two active sites, which are distal from the dimer interface. Assignment of backbone NMR resonances and engineering of a redox switch show that two helices position a loop containing catalytic residues within each active site.

Binding Sites↗
PubMed2004

Proteins of purified Epstein-Barr virus.

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

Capsid Proteins↗