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PubMed2004

Functional analysis of virion host shutoff protein of pseudorabies virus.

During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.

Amino Acid Sequence↗
PubMed1970

Effect of spermine on host-cell lysis and reproduction by a lactic streptococcal bacteriophage.

A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

Agar↗
PubMed1975

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.

Adenoviridae↗
PubMed1992

Significance of the detection of Epstein-Barr virus DNA in lymph nodes in patients with Hodgkin's disease.

Epstein-Barr virus (EBV) DNA is frequently identified in benign and malignant lymphoproliferative conditions. As shown by in situ hybridization studies viral DNA is localized within malignant cells as well as benign lymphocytes. Clonal and nonclonal EBV genomes are present in Hodgkin's disease (HD), lymphomas of the immunocompromised host and reactive lymph node hyperplasia. Lytic infection with formation of linear genomes is observed in the same conditions but appears to be infrequent in HD as shown by quantitation of mRNA coding for viral capsid antigen. Expression of the oncogene LMP (latent membrane protein) is seen in Sternberg-Reed (SR) cells and immunoblasts of AIDS-related lymphoma and infectious mononucleosis (IM). In HD, the region of the BNLF1 oncogene coding for the amino terminal and transmembrane domains (associated with oncogenic function) of LMP appears to be homogeneous whereas the region coding for the intracytoplasmic (carboxy terminal) domain of LMP is heterogeneous. Cytological similarities between SR cells and immunoblasts of IM and AIDS-related lymphomas are consistent with the hypothesis that the BNLF1 oncogene is one possible inducer of morphological features of SR cells. Whether chromosomal integration of EBV DNA is an important factor in activation of such a transforming activity remains to be elucidated. EBV DNA positive and negative HD cases with numerous SR cells lack significant mRNA expression of the two recombinase activating genes (RAG-1 and RAG-2). Therefore the SR cells appear to be derived from lymphocytes beyond the pre-B-cell or common thymocyte stage which may or may not subsequently become infected by EBV.

Antigens, Viral↗
PubMed1990

Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly.

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Chromosome Deletion↗
PubMed2004

Enhancement of correct protein folding in vivo by a non-lytic baculovirus.

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

Animals↗
PubMed2002

Pathogenesis of herpes simplex virus type 2 virion host shutoff (vhs) mutants.

During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.

Animals↗
PubMed1995

Immunity determinant of phage-plasmid P4 is a short processed RNA.

In the phage-plasmid P4, both lysogenic and lytic functions are coded by the same operon. Early after infection the whole operon is transcribed from the constitutive promoter PLE. In the lysogenic condition transcription from PLE terminates prematurely and only the immunity functions, which are proximal to the promoter, are thus expressed. Fragments of the P4 immunity region were cloned in an expression vector. A DNA fragment as short as 91 bp was sufficient, when transcribed, to express P4 immunity and to complement P4 immunity deficient mutants. This fragment, like prophage P4, produced a 69 nt long RNA (CI RNA). A shorter P4 fragment neither expressed immunity nor synthesized the CI RNA. Thus the CI RNA is the P4 trans-acting immunity factor. The 5' end of the CI RNA, mapped by primer extension, does not correspond to the transcription initiation point, thus suggesting that the CI RNA is produced by processing of the primary transcript. In an RNase P mutant host the processing of the 5' end and the production of a functional CI RNA were impaired. The requirement of RNase P for the correct processing of CI appears to be related to the predicted secondary structure of the precursor CI RNA. A region (seqB) within the CI RNA shows complementarity with two cis-acting sequences (seqA and seqC) required for P4 immunity, suggesting that transcription termination may be caused by pairing of the CI RNA with the complementary target sequences on the nascent transcript.

Base Sequence↗
PubMed1995

The products of gene I and the overlapping in-frame gene XI are required for filamentous phage assembly.

The class I filamentous bacteriophage are non-lytic single-stranded DNA phage, which are assembled at the cell envelope as they are extruded from the Gram-negative bacteria, Escherichia coli. The process requires the products of the phage genes I and IV, which reside in the inner and outer membrane, respectively, and are not present in the mature phage particle. Gene I encodes two proteins, the full length 348-residue pI and a smaller pI*, which this report shows is the result of an internal translation initiation event at methionine codon 241. Both pI and pI* are shown to be required for phage assembly. Therefore, pI* can be considered the product of an additional phage gene, XI, which is a separate in-frame gene that overlaps gene I. Both proteins contain a 13-residue region adjacent to the cytoplasmic face of the inner membrane that probably exists as a positively charged amphiphilic helix. Although this region is not required for membrane insertion of pI and pI*, it is shown to be required for phage assembly. Oligonucleotide-directed mutagenesis of this region, which removes positive charges or alters the hydrophobic face of the putative helix, renders pI and pI* unable to function in phage assembly. This region of pI and pI* is highly homologous in structure to the carboxyl-terminal 11 amino acids of pVIII, the main coat protein, which also reside adjacent to the cytoplasmic face of the inner membrane.

Amino Acid Sequence↗
PubMed2000

Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis.

Streptococcus thermophilus is a lactic acid bacterium used in industrial milk fermentation. To obtain phage-resistant starters, S. thermophilus strain Sfi1 was submitted to mutagenesis with the thermolabile insertional vector pG(+)host9:ISS1 followed by a challenge with the lytic S. thermophilus phage Sfi19. Vector insertions into four distinct sites led to a phage-resistance phenotype. Three mutants were characterized further. They were protected against the homologous challenging phage and 14 heterologous phages. All three mutants adsorbed phages. No intracellular phage DNA synthesis was observed in mutants R7 and R71, while mutant R24 showed a delayed and diminished phage DNA synthesis compared to the parental Sfi1 strain. In mutant R7 a short deletion occurred next to the insertion site which removed the upstream sequences and the 15 initial codons from orf 394, encoding a likely transmembrane protein. Analogy with other phage systems suggests an involvement of this protein in the phage DNA injection process. In mutant R24 the vector was inserted into orf 269 predicting an oxido-reductase. When the vector sequence was removed via homologous recombination across the duplicated insertion elements, mutant R24 returned to the phage susceptibility of the parental strain. This observation suggested that inactivation of orf 269 was not crucial for the resistance phenotype. A gene encoding a likely restriction subunit of a type I restriction-modification system was located directly downstream of the insertion site in mutant R24. hsdM and hsdS genes encoding the modification and specificity subunits of a type I R-M system and biological evidence for an active R-M system were detected in strain Sfi1, suggesting involvement of a type I R-M system in the resistance phenotype of R24.

Amino Acid Sequence↗
PubMed1985

Effect of tacaribe virus infection on host cell protein and nucleic acid synthesis.

Tacaribe virus stocks were prepared which induced definite lytic responses in Vero cells infected at multiplicities giving synchronous infection. Under these conditions, the first signs of cytopathic effect (c.p.e.) appeared at about 30 h post-infection and cell lysis occurred after 40 h. Before the onset of cytopathic changes, the virus induced inhibition of host cell protein, DNA and RNA (primarily rRNA) synthesis. These were designated c.p.e. (+) virus stocks. The effect of virus on host cell macromolecular synthesis and development of c.p.e. were not related to the virus isolate, but to the conditions under which the virus was produced. Thus, from a single virus clone, working stocks were derived which could or could not induce inhibition of host cell functions and c.p.e. development. The virus stocks that did not induce inhibition are defined as c.p.e. (-). Analysis of [3H]leucine-labelled proteins from Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks revealed synthesis of two virus-specific polypeptides migrating with mobilities corresponding to mol. wt. 68 000 and 79000. These are presumed to correspond, respectively, to the nucleoprotein and to the minor polypeptide p79. In cells infected with the c.p.e. (+) virus stock, the virus-specific polypeptides were synthesized at times when there was a drastic inhibition of host cell protein synthesis. The yield of infectious progeny during the first 24 h of infection is similar in Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks. The proportion of defective interfering particles was much higher in the c.p.e. (-) than in the c.p.e. (+) virus stocks. The results presented here are the first demonstration that an arenavirus affects the biosynthetic machinery of the host cell.

Animals↗
PubMed1996

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.

Blotting, Northern↗
PubMed2003

Productive infection of cerebellar granule cell neurons by JC virus in an HIV+ individual.

BACKGROUND: In the setting of severe immunosuppression, the polyomavirus JC (JCV) can cause a lytic infection of oligodendrocytes. This demyelinating disease of the CNS white matter (WM) is called progressive multifocal leukoencephalopathy (PML). JCV has a very narrow host-cell range and productive infection of neurons has never been demonstrated. Patient, methods, and results: An HIV-1-infected patient presented with signs of pyramidal tract and cerebellar dysfunction. Brain MRI revealed T2 hyperintensities in the WM of both frontal lobes and cerebellar atrophy. His disease progressed despite therapy and he died 6 months later. In addition to classic PML findings in the frontal lobe WM, autopsy revealed scattered foci of tissue destruction in the internal granule cell layer (IGCL) of the cerebellum. In these foci, enlarged granule cell neurons identified by the neuronal markers MAP-2 and NeuN reacted with antibodies specific for the polyomavirus VP1 capsid protein. Electron microscopy showed 40 nm viral particles, consistent with polyomaviruses, in these granule cell neurons. In addition, JCV DNA was detected by PCR after laser capture microdissection of cells from the areas of focal cell loss. Finally, in situ hybridization studies demonstrated that many granule cell neurons were infected with JCV but did not contain viral proteins. Sequence analysis of the JCV regulatory region from cerebellar virions showed a tandem repeat pattern also found in PML lesions of the frontal lobe WM. CONCLUSION: JCV can productively infect granule cell neurons of the IGCL of the cerebellum. This suggests a role for JCV infection of neurons in cerebellar atrophy occurring in HIV-infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Type I consensus IFN (IFN-con1) gene transfer into KSHV/HHV-8-infected BCBL-1 cells causes inhibition of viral lytic cycle activation via induction of apoptosis and abrogates tumorigenicity in sCID mice.

In this study, we investigated the effects of human type I consensus interferon (IFN-con1) (Amgen) gene transfer into body cavity-based lymphomas (BCBL)-1 cells, which are latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV) human herpesvirus-8 (HHV-8). Both the basal and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated production of KSHV/HHV-8 mature virions was strongly inhibited in genetically modified IFN-producing BCBL-1 cells as compared with parental or control transduced counterparts. A similar inhibition was obtained on treatment of parental BCBL-1 cells with exogenous IFN-con1. The reduction in KSHV/HHV-8 production was associated with a decrease in the basal and TPA-stimulated intracellular amount of the linear form of the viral genome. Interestingly, 25%40% of the IFN-producing BCBL-1 cell population underwent spontaneous apoptosis in vitro. TPA treatment, which did not significantly affect the viability of the parental and control BCBL-1 cells, resulted in the apoptotic death of up to 70% of the IFN-producing cell population. Addition of exogenous IFN-con1 to parental BCBL-1 cells produced similar effects, although less intense. Injection of either parental or control-transduced BCBL-1 cells into SCID mice resulted in progressively growing tumors characterized by an unusually high level of tumor angiogenesis. In contrast, complete tumor regression was observed in all the mice injected either subcutaneously (s.c.) or intraperitoneally (i.p.) with the IFN-producing BCBL-1 cells. These results represent the first evidence that type I IFN can counteract the activation of a productive herpesvirus infection in latently infected tumor cells by the induction of apoptosis, providing an interesting link between the antiviral and antitumor activities of this cytokine. These data suggest the possible advantages of strategies of type I IFN gene transfer (with respect to the use of the exogenous cytokine) for the treatment of patients with some HHV-8-induced malignancies.

Animals↗
PubMed2006

Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B7-1 in an immunocompetent murine model.

PURPOSE: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy. EXPERIMENTAL DESIGN: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-gamma enzyme-linked immunospot assay, and immunohistochemical studies. RESULTS: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-gamma levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4(+) and CD8(+) T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7. CONCLUSION: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.

Adenoviridae↗
PubMed1993

Transcripts from the Epstein-Barr virus BamHI A fragment are detectable in all three forms of virus latency.

An unexpected feature of the latency II form of Epstein-Barr virus (EBV) infection seen in the epithelial tumor nasopharyngeal carcinoma (NPC) is the presence of spliced polyadenylated RNAs encoded from the BamHI A fragment of the viral genome and running in the opposite orientation to several BamHI-A lytic cycle genes. The importance of these BamHI-A transcripts and the specificity of their association with NPC remain to be determined. In this study, we examined the extent to which such RNAs are present in other transcriptionally distinct forms of EBV latency seen in B cells. Two independent assays of BamHI-A transcription were employed: amplification across defined splice junctions in cDNAs, using the polymerase chain reaction, and in situ hybridization with a radiolabeled riboprobe specific for a putative open reading frame downstream of these splice junctions. Such methods, which easily detected BamHI-A RNAs in fresh NPC biopsies and transplantable NPC lines, also revealed consistent expression of these transcripts in all EBV-positive Burkitt's lymphoma cell lines displaying the highly restricted latency I form of infection (BamHI-F promoter usage) as well as in all EBV-transformed lymphoblastoid cell lines (LCLs) displaying the latency III form of infection (BamHI-C/W promoter usage). Expression in established LCLs, occurring irrespective of virus producer status, was not a consequence of continued in vitro passage; thus, appropriately spliced BamHI-A transcripts could be amplified from normal B cells within 1 day of their experimental infection in vitro, along with BamHI-C/W promoter-initiated but not BamHI-F promoter-initiated mRNAs. In situ hybridization both on Burkitt's lymphoma cell lines and on LCLs showed that essentially every cell contained BamHI-A transcripts, although at levels apparently lower than those observed in NPC. We conclude that expression of the BamHI-A RNAs is a consistent feature shared by all known forms of latent EBV infection.

Animals↗
PubMed2001

Kinetic analysis of the steps of the polyomavirus lytic cycle.

Kinetic studies of the accumulation of early and late transcripts, early and late proteins, genomes, and live virus, during the lytic cycle of murine polyomavirus wild-type A2, were carried out in synchronized NIH 3T3 cells released from G(0) by the addition of serum after infection. This first-time simultaneous analysis of all parameters of the virus life cycle led to new insights concerning the transcriptional control at the early-to-late transition. During the early phase, early transcripts were synthesized at very low levels, detectable only by reverse transcription-PCR, from 6 h postinfection (hpi). Large T protein could be detected by 8 hpi (while infected cells were in the G(1) phase). The level of expression of the middle T and small T proteins was lower than that of large T at all times, due, at least in part, to a splicing preference for the large-T 5' splice site at nucleotide 411. A large increase in the level of both early and late transcripts coincided closely with the detection in mid-S phase of viral genome amplification. Thereafter, both classes of transcripts continued to further accumulate up to the end of the experiments (48 hpi). In addition, during the late phase, "giant" multigenomic transcripts were synthesized from the early as well as the late promoter. Thus, a major type of transcriptional control appears to be applied similarly to the transcription of both early and late genes. This view differs from that in the literature, which highlights the enhancement of late transcription and the repression of early transcription. However, despite this parallel transcriptional control, additional regulations are applied which result in higher levels of late compared to early transcripts, as previously described. In the accompanying article, a key role for middle T and/or small T in this late-phase enhancement of early and late transcription is demonstrated (16). Other novel findings, e.g., the synthesis of a very abundant short early promoter proximal RNA, are also described.

3T3 Cells↗
PubMed2003

Polyomavirus hr-t mutant-specific induction of a G2/M cell-cycle arrest that is not overcome by the expression of middle T and/or small T.

The polyomavirus hr-t class of mutants has served as a major prototype to study the function of middle T + small T in the virus lytic cycle, Biochem. Biophys. Acta 695 (2), 69-95). The properties of these middle T + small T defective mutants were defined by comparisons with "wild-type" strains reconstructed by marker rescue. Similar comparisons in the A2 genetic background have revealed a number of differences, J. Virol. 75, 8380-8389). Here we describe a major divergence in their effects on cell-cycle progression of both permissive mouse NIH3T3 cells and semipermissive Fischer rat FR3T3 cells. Infection of NIH3T3 or FR3T3 cells in serum-rich medium with wild-type A2 (WTA2) or WTA2-derived middle T + small T-defective mutants did not perturb cell cycling, tested up to entry into the third cycle. In contrast, infection with four hr-t mutants analyzed, examined in detail with mutant B2, resulted in an accumulation of cells in G2/M in a dose-dependent and serum-independent manner. The arrest began in the first cell cycle. At multiplicities of infection above 10 PFU/cell, 50-80% of the cell population became arrested by the end of the second cycle. FR3T3 arrested cells detached from the monolayer with a rounded up morphology. Three other hr-t mutants investigated were also found to arrest cells in G2/M. Expression of middle T and/or small T either in trans or in cis did not abrogate this cell-cycle arrest, as demonstrated in the latter case with the middle T + small T expressing strain "wtB2" obtained by repair of the B2 deletion. In FR3T3 cells, the induction of a cell-cycle arrest by wtB2 was accompanied by a severe delay and reduction in neoplastic transformation relative to WTA2 used at equal dose. Mutation(s) in the C-terminal domain of large T antigen, upstream of the site-specific DNA binding activity, is necessary for the cell-cycle block. The possible causes for the cell-cycle block are discussed.

3T3 Cells↗