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PubMed1988

Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB.

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

Amino Sugars↗
PubMed2005

Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production.

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAV(UK) viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.

Active Transport, Cell Nucleus↗
PubMed1998

Variations in gene expression and genomic stability of human hepatoma cells integrated with hepatitis B virus DNA.

The association of hepatitis B virus (HBV) infection with human hepatoma is well established. However, no consensus regarding the etiology of hepatocellular carcinoma was elucidated. In this paper, the genomic stability and gene expression of HBV DNA-integrated and non-integrated hepatoma cells by Transcript Profile (TP)-PCR and RT-PCR are characterized. The additional DNA bands generated from TP-PCR of HBV integrated genomes were not correlated with the sequence of HBV, suggesting that the variations may result from genomic instability of the host cells. Moreover, differential genes expressed in HBV DNA-integrated cells were sequenced. A cDNA generated from the integrated cells exhibited 99.3% homology with the sequences of ATP synthase 6 and cytochrome C oxidase III, but the sequences were abnormally linked together. Since HBV infection may alter the energy metabolism of the cell, the results suggest that the integration may cause mitochondriae defects in the ATP synthase 6 and cytochrome C oxidase III genes.

ATP Synthetase Complexes↗
PubMed1985

Relationship between serum and histochemical markers for hepatitis B virus and rate of viral integration in hepatocellular carcinomas in Japan.

The relationship between serological and histochemical markers of hepatitis B virus (HBV) and viral integration in hepatocellular carcinomas (HCCs) in Japan was investigated. Special attention was paid to the exclusion of false-positive results due to bacterial contamination of autopsy materials. Of 85 patients with HCC, 23 were positive for serum HBsAg, 18 were positive for serum HBV antibodies and the remaining 44 were negative for serum HBV markers. Among the 23 HCCs from HBsAg carriers, 19 (82.6%) had integrated HBV DNA in the tumor DNA. In contrast, of the 18 HCCs from HBV antibody-positive patients, only one was positive for HBV DNA integration; this particular HCC was from a long-term HBsAg carrier who became a non-carrier by seroconversion at a later age. Of 44 HCCs from patients negative for serum HBV markers, 3 had integrated HBV DNA. Of these 3 cases, 2 were, however, histochemically positive for HBsAg in non-cancerous portions of the liver. Thus, our present results show a high integration rate of HBV DNA in HCCs of carrier patients and an extremely low rate of viral integration in HCCs of non-carrier patients. The latter finding contrasts strongly with previous results gained from analysing materials from patients in Europe and Africa. No histological differences were apparent between HCCs with or without viral integration.

Antibodies, Viral↗
PubMed1988

Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein.

The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.

Base Sequence↗
PubMed2001

Integration of adeno-associated virus 2 DNA in human MKR melanoma cells induces a peptide with oncosuppressive properties.

Integration of adeno-associated virus type 2 (AAV2) DNA into the genome of the human MKr melanoma cell line grossly alters the cellular phenotype of these cells. Cultures derived from AAV DNA-harboring single cells share various similarities with normal cells in culture, and a considerable number of clones show signs of cellular senescence and terminal differentiation. Medium conditioned by such terminally differentiating cells contains a small cytokine-like factor (AAV-induced factor [AIF]). The factor was characterized as peptide, which influences cell adhesion and appears to exist in forms of different activity connected by a trypsin-like cleavage site. It modulates cell growth in opposite ways: proliferation of tumor-derived cells is inhibited and fibroblasts are stimulated. The observations suggest that integration of AAV may result in cytokine induction and thus can indirectly affect growth of distant AAV-free target cells in a paracrine or endocrine manner. This is of interest with regard to anti-oncogenic properties of AAV and may also imply the possibility of AAV pathogenicity.

Antineoplastic Agents↗
PubMed2002

Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I digestion.

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8.5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.

Cell Nucleus↗
PubMed2004

Integration of Epstein-Barr virus into chromosome 6q15 of Burkitt lymphoma cell line (Raji) induces loss of BACH2 expression.

Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is a well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50-60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly guanine/cytosine-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent host sequences. EBV was present at intron 1 of the BACH2 gene located on chromosome 6q15. BACH2 was not expressed in the Raji cell line. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis.

Animals↗
PubMed1996

Viral integration sites in human papilloma virus-33-immortalized cervical keratinocyte cell lines.

The viral organization of HPV-33 was determined by Southern blotting in 2 HPV-33-immortalized cervical cell lines (CK11 and CK12) and compared to our previous results obtained on 10 other already characterized HPV-33-immortalized cell lines (CK1 to CK10). As observed in CK1 to CK10, the viral DNA was found integrated in the cellular genome of CK11 and CK12. However, in CK11 and CK12, the integrated viral genome was deleted and mostly limited to the URR and the E6-E7 ORFs, stressing the importance of those sequences in the immortalization process. Furthermore, CK11 and CK12 showed a unique and identical integration site, as observed in CK1 to CK10, which also harbored HPV-33 integrated at a unique and identical site (which was however different from the one evidenced in CK11 and CK12). Indeed, in situ hybridizations on chromosomes allowed the precise localization of the viral DNA on chromosome 13q33-34 in CK1 to CK10 whereas it was mapped to chromosome 9p13 in CK11 and CK12. We discuss the possibility that integration of HPV-33 at those two particular sites has conferred some growth advantages to the cells and could have thus played a crucial role in the immortalization.

Cell Line, Transformed↗
PubMed1996

Adult T-cell leukaemia/lymphoma with multiple integrations of human T-cell lymphotropic virus type I proviral DNA: differing clinical features are linked to varied proviral integration.

Multiple integrations of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA are occasionally found in tumor cells from patients with adult T-cell leukaemia/lymphoma (ATL). However, the clinical implications of multiple integrations of HTLV-I in ATL have not been well established. We studied 95 patients with ATL to elucidate the relationship between the multiple integrations of HTLV-I and the clinical characteristics. The proviral DNA of HTLV-I was examined by standard Southern blot analysis using the probe of an entire HTLV-I genome and the endonucleases with or without cleavage sites within the provirus. Multiple integrations of HTLV-I were detected in eight patients as extraordinary multiple bands; five patients showed multiple bands of the same intensity, and the remaining three showed multiple bands of differing intensities. The patients were divided into two groups based on these band patterns. One group was considered to exhibit one tumour cell clone carrying multiple copies of the provirus, whereas the other was considered to exhibit multiple tumour cell clones, each carrying one copy of the provirus. The former group of patients manifested a highly aggressive clinical course with frequent peculiar organ infiltrations, including the retina, uvea and muscle, along with the presence of large peripheral leukaemic T cells having flower-like nuclei. The latter group demonstrated an indolent clinical course with skin lesions or small leukaemic T cells having cleaved or lobulated nuclei. These findings suggest that the pattern of multiple HTLV-integrations into the tumor cell(s) has clinical implications in ATL. This may help to explain the heterogeneity of this disease.

Adult↗
PubMed1988

Microdeletion associated with the integration process of hepatitis B virus DNA.

Hepatitis B virus (HBV) DNA is often found in integrated form in hepatocellular carcinomas (HCC) and in non-cancerous liver cells of chronic carriers of HBV. However, the process of integration has not been well understood. Analyses of integrant DNA was expected to give clues. However, the majority of the integrants are products of multistep rearrangements following integrations, and analysis of randomly selected samples do not give clues for understanding the process of primary integrant formation. Therefore, one must select an appropriate integrant(s) that has a simple structure. We surveyed a collection of integrants prepared from many HCC's, and found one integrant that has the simplest structure so far studied: The viral genome is almost complete, is joined to cellular DNA using the cohesive end of the viral DNA, and furthermore, the "left" and "right" flanking cellular DNA's are almost contiguous. Analysis of the unoccupied sites in cellular DNA showed that, although almost contiguous, it has generated a microdeletion (15 base pairs) in the target sequence. This target sequence has a short region of homology to the sequence in the viral genome located close to the junction. One integrant with strikingly similar features has been reported independently. Two similar, but not identical cases from literatures could be added to this category. Therefore, the integrants with these properties may represent a unique category among those prepared from hepatocellular carcinomas. Based on these findings, we propose that this integrant represents the primary product of integration, and discuss the intermediate acting in the process of integration.

Base Sequence↗
PubMed1993

Reversion of a polymerase-defective integrated HIV-1 genome.

The 8E5 clonal cell line, derived from HIV-1-infected CEM cells, carries a single, reverse transcriptase (RT)-defective copy of an integrated HIV genome. The absence of RT production is a consequence of a frame shift in the pol gene, due to the addition of a single base at position 3241. We report here that 8E5 cells produce an infectious virus that can be serially passaged on CD4+ lymphoid cells. This virus (8E5R) is RT positive, but displays a slow replication profile, together with a reduced cytopathic effect. The nucleotide sequence of a segment of the pol region produced by PCR amplification of DNA from 8E5R-infected cells shows that the single nucleotide insertion characteristic of the 8E5 genome had been corrected. The same reversion event was also found to occur in most single-cell clones derived from the 8E5 cell line. Because this cell line is used in many laboratories, notably as a standard for PCR quantitation, and is generally considered as unable to produce infectious virus, our findings should prompt investigators to use particular care in the handling of these cells.

Base Sequence↗
PubMed2001

Characterization of adenovirus-induced inverted terminal repeat-independent amplification of integrated adeno-associated virus rep-cap sequences.

Stable packaging cell lines expressing the rep and cap genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa rep-cap cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the inverted terminal repeats (ITRs) deleted. We now report a more detailed analysis of this phenomenon and highlight the key cellular and viral factors involved. Determination of the rep-cap copy number of HeRC32 cells indicated that maximum rep-cap amplification occurred between 24 and 48 h following adenovirus infection. Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified rep-cap sequences were found in an extrachromosomal form. Amplification of the rep-cap sequence with the ITRs deleted was not dependent on adenovirus replication and still occurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indicating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was essential, since rep-cap amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant encoding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of rep-cap amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in rep-cap amplification. Indeed, the lack of detectable amplification in an adenovirus-infected stable rep-cap HeLa cell clone unable to produce Rep proteins further supported that, among the viral gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated rep-cap sequences in the absence of the AAV ITRs.

Adenoviridae↗
PubMed1992

Structure and expression of human Fli-1 gene.

Three ets family members, v-ets, spleen focus forming virus proviral integration 1/Pu.1, and Friend leukemia integration 1 (Fli-1), were shown to be involved in retroviral mediated acute leukemias suggesting that ets family members play a crucial role in transformation. Mouse Fli-1 was shown to be involved in 75% erythroleukemias induced by Friend murine leukemia virus suggesting the possibility that Fli-1 may play a critical role in cellular transformation. Since Fli-1 maps to the mouse chromosome region syntenic with human chromosome 11q23-24, it is tempting to speculate that human Fli-1 may be involved in human sarcomas, leukemias, and lymphomas involving human chromosome 11q23-24. We have isolated complementary DNA clones representing the human homologue of Fli-1 gene. Nucleotide sequence analysis revealed that the human Fli-1 gene codes for a 452-residue protein the predicted amino sequence of which shows 80% homology to the human erg-2 protein previously described. A 3.5-kilobase transcript of the human Fli-1 gene was observed in different cells. Sequence analysis revealed two domains of ets homology, one at the 5' and the other at the 3' end of the Fli-1 gene. This 3'-ets homology domain, which is mainly responsible for DNA binding activity, is seen in all the ets family members; however, the 5'-ets homology region is conserved in only five genes, Fli-1, c-ets-1, ets-2, GABP-alpha, and erg, suggesting a common biological function which is shared among these genes. Interestingly, mouse and human Fli-1 transcripts contain highly homologous 5'-untranslated region suggesting that this conserved region may play an important role in the posttranscriptional regulation of the Fli-1 transcript.

Amino Acid Sequence↗
PubMed1995

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.

Cell Line↗
PubMed1979

The influence of arginine starvation on the synthesis of virus high molecular weight DNA in HeLa cells productively infected by adenovirus type 5.

In culture cells productively infected by adenovirus a high mol. wt. form of DNA is synthesized which is known to represent, at least in part, virus DNA integrated into cellular DNA. We found that the synthesis of this high mol. wt. DNA and the other DNA size classes can strongly and differentially be influenced by altering the metabolic state of the cells. The effects of different rates of cell growth were tested in this respect as well as arginine deprivation as opposed to application of complete growth medium. Synthesis of virus high mol. wt. DNA and unit genome length DNA is enhanced in actively growing as compared to resting Ad5-infected HeLa cells. Under arginine deficiency, in resting Ad5-infected HeLa cells, integration of virus DNA sequences into cellular DNA is almost totally suppressed whereas virus unit genome length DNA is still synthesized. This differential effect is interpreted by the hypothesis that the formation of virus high mol. wt. DNA is a synthetic process that is independent of the unit size virus DNA replication, but coupled to the synthesis of a special form of a special form of cellular DNA that is less effectively shut off by the infection than cellular DNA in general.

Adenoviruses, Human↗