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At least 793 records · Page 44Linked to original sources
PubMed2026

Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking machinery as a potential target for developing antiviral strategies.

Animals↗
PubMed1998

Expression analysis of recombinant herpes simplex virus type 1 DNase.

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.

Animals↗
PubMed1992

Intramolecular homologous recombination in Bacillus subtilis 168.

Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B. subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene. Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions. These results are consistent with the hypothesis that B. subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.

Bacillus subtilis↗
PubMed2005

Viruses - seeking and destroying the tumor program.

DNA viruses have enormous utility in cancer research, both as tools for tumor target discovery as well as agents for lytic cancer therapies. This is because there is a profound functional overlap between the DNA viral and tumor cell programs. DNA viruses encode proteins that elicit growth deregulation in infected cells similar to that engendered by mutations in tumor cells. Evolution has refined viral proteins to target the critical cellular hubs that regulate growth. Thus, viral proteins are discriminating biochemical probes that can be used to identify and characterize novel tumor targets. Moreover, the overlap between the DNA viral and tumor programs can also be exploited for the development of lytic cancer therapies. Discovering whether tumor cells selectively complement the replication of viral mutants can reveal novel oncolytic viral therapies, as well as unexpected tumor properties. For example, altered RNA export was recently uncovered as a novel tumor cell property that underlies ONYX-015 replication, a promising oncolytic adenoviral therapy. A perspective is provided on how adenovirus could be systematically exploited to map the requisite role, or indeed the redundancy, of cellular pathways that act in an integrated program to elicit pathological replication. This knowledge has important applications for the rational design of the next generation of oncolytic viruses, as well as the discovery of efficacious combination cancer therapies.

Adenoviridae↗
PubMed2000

Postexposure vaccination massively increases the prevalence of gamma-herpesvirus-specific CD8+ T cells but confers minimal survival advantage on CD4-deficient mice.

Mice that lack CD4(+) T cells remain clinically normal for more than 60 days after respiratory challenge with the murine gamma-herpesvirus 68 (gammaHV-68), then develop symptoms of a progressive wasting disease. The gammaHV-68-specific CD8(+) T cells that persist in these I-A(b-/-) mice are unable to prevent continued, but relatively low level, virus replication. Postexposure challenge with recombinant vaccinia viruses expressing gammaHV-68 lytic cycle epitopes massively increased the magnitude of the gammaHV-68-specific CD8(+) population detectable by staining with tetrameric complexes of MHC class I glycoprotein + peptide, or by interferon-gamma production subsequent to in vitro restimulation with peptide. The boosting effect was comparable for gammaHV-68-infected I-A(b-/-) and I-A(b+/+) mice within 7 days of challenge, and took more than 110 days to return to prevaccination levels in the I-A(b+/+) controls. Although the life-span of the I-A(b-/-) mice was significantly increased, there was no effect on long-term survival. A further boost with a recombinant influenza A virus failed to improve the situation. Onset of weight loss was associated with a decline in gammaHV-68-specific CD8(+) T cell numbers, though it is not clear whether this was a cause or an effect of the underlying pathology. Even very high levels of virus-specific CD8(+) T cells thus provide only transient protection against the uniformly lethal consequences of gammaHV-68 infection under conditions of CD4(+) T cell deficiency.

Animals↗
PubMed1974

Sequence arrangements in clonal isolates of polyoma defective DNA.

Five clonal isolates of purified polyoma defective DNA [Fried, M. (1974) J. Virol. 13, 939-946] have been examined by electron microscopy. Four isolates (D-92, D-74, D-50, and D-47) are largely homogeneous in sequence, whereas, the fifth isolate (D-80) is somewhat heterogeneous.Polyoma nondefective DNA is cleaved in a unique region of the genome by the EcoR(I) endonuclease. Hybridization of the resulting linear molecules with randomly nicked defective DNA reveals distinguishable types of heteroduplex structures for each of the different defective DNAs. Although the defective DNAs are shorter than polyoma nondefective DNA, the heteroduplex experiments demonstrate that they are not simply deletion mutants containing only a portion of the viral genome. Three isolates (D-92, D-50, and D-47) contain regions of homology to polyoma DNA covalently linked to non-homologous regions. One isolate (D-74) contained no regions of detectable homology to polyoma DNA. Another isolate (D-80) contained a large proportion of molecules with duplicated-inverted regions. Some of these isolates of defective DNA may contain specific host sequences at the site(s) of integration of the polyoma genome during the lytic cycle in mouse cells. A process we term "abortive replication" may explain the formation of different types of defective DNAs.

Base Sequence↗
PubMed1977

State of prophage Mu DNA upon induction.

We have compared the process of prophage lambda induction with that of prophage Mu. According to the Campbell model, rescue of lambda DNA from the host DNA involves reversal of lambda integration such that the prophage DNA is excised from the host chromosome. We have monitored this event by locating the prophage DNA with a technique in which DNA of the lysogenic cells is cleaved with a restriction endonuclease and fractionated in agarose gels. The DNA fragments are denatured in gels, transferred to a nitrocellulose paper, and hybridized with 32P-labeled mature phage DNA. The fragments containing prophage DNA become visible after autoradiography. Upon prophage lambda induction, the phage-host junction fragments disappear and the fragment containing the lambda att site appears. No such excision is seen in prophage Mu. The Mu-host junction fragments remain intact well into the lytic cycle, when Mu DNA has undergone many rounds of replication and apparently many copies of Mu DNA have been integrated into the host DNA. Therefore, we postulate that Mu DNA replicates in situ and the replication generates a form of Mu DNA active in the integrative recombination between Mu DNA and host DNA. This type of mechanism may be common to many transposable elements.

Coliphages↗
PubMed2006

In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium.

The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.

Animals↗
PubMed1990

Characterization of the C operon transcript of bacteriophage Mu.

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.

Bacteriophage mu↗
PubMed1978

Flow microfluorometric analysis of herpesvirus infected BHK-21 and BALB/3T3 cell cultures.

The interaction of herpes simplex type 1 with two eukaryotic cell lines (BHK-21 and BALB/3T3) was investigated by flow microfluorometric analysis after cell staining with mithramycin. Uninfected, lytically infected and persistently infected cell populations were examined. Viral replication within an infected cell population could be detected via FMF analysis. The low levels of viral replication occurring within persistently infected cell populations were also detectable. A marked degree of correlation was noted between morphological observations of infected cultures and the FMF profiles obtained. The results deomonstrate the great potential of this technique for the early detection and analysis of viral infection.

Cell Count↗
PubMed2003

Functional analysis of the mutated Epstein-Barr virus oncoprotein LMP1(69del): implications for a new role of naturally occurring LMP1 variants.

BACKGROUND AND OBJECTIVES: The role of carboxyterminal deletions of the latent membrane protein-1 (LMP1) in Epstein-Barr virus (EBV) infection and oncogenesis is unclear. Here we describe functional properties of a rare 69-bp LMP1 deletion mutant (LMP1(69del)) isolated from a patient with polyclonal B-cell lymphocytosis. DESIGN AND METHODS: Colony focus assay was used to evaluate the transforming capacity of LMP1(69del) in comparison to that of wild-type LMP1 from EBV strain B95/8. Transient transfectants of B-, T-, epithelial and 3T3 cells, and stable transfectants with ecdysone-inducible LMP1 expression were produced. The signaling capacity of both LMP1s on nuclear transcription factors NFkappaB and AP-1 were studied. Secretion of matrix metalloproteinase MMP-9, apoptosis, and EBV lytic and latent gene expression were also investigated. RESULTS: LMP(69del) showed transforming properties comparable to those of the wild-type oncoprotein. Induction of NFkappaB but a markedly reduced influence on AP-1 were observed. Both oncoproteins induced secretion of MMP-9, and enhanced pre-apoptotic effects in Jurkat-T cells leading to increased Fas/Apo-1 and doxorubicin-mediated apoptosis. Furthermore, LMP1(69del) showed a more effective down-regulation of the EBV lytic cycle master gene BZLF1(Zebra) than did wild-type LMP1. INTERPRETATION AND CONCLUSIONS: (i) LMP1(69del) possesses oncogenic properties, (ii) the observed impaired activity on AP-1 does not interfere with MMP-9 induction, (iii) the enhanced inhibition of BZLF1 could compensate for previously described mutations of our isolate leading to a more lytic phenotype and may be responsible for counteracting permanent virus replication in the chronic active EBV syndrome observed in this patient.

Animals↗
PubMedDate not supplied

Transposition without duplication of infecting bacteriophage Mu DNA.

Most models of DNA transposition invoke replication of the transposable element, but it is not clear whether a 'co-integrate' is an obligatory intermediate in the pathway leading to the production of simple insertions during transposition. Such an intermediate can be accounted for only by a replicative transposition scheme. Bacteriophage Mu is a temperate phage that can either lysogenize or lyse its host, and it encodes at least two modes of transposition as judged by the end-products generated by the process. During the lytic development of the integrated prophage, co-integrates are the predominant end-products; transposition is coupled to replication during this phase. A small number of simple insertions are also produced during the lytic growth, but during transposition from the infecting phage into the host chromosome, simple insertions are the main end-products. Conditions can be found where the choice between the two kinds of end-products depends on a delicate balance between the essential transposition functions encoded by Mu. Experiments have suggested that the simple insertions which arise during transposition from the infecting phage may do so without Mu DNA replication. Here I demonstrate using an infecting phage with completely methylated DNA, a dam- (DNA adenine methylase) host and a combination of restriction enzymes that can cut either fully methylated or unmethylated DNA but not hemi-methylated DNA, that transposition of the phage DNA into the host chromosome does not involve a duplication of its DNA. This result may also have significance for other transposons that do not appear to go through a co-integrate intermediate during transposition.

Bacteriophage mu↗
PubMed1995

Kid, a small protein of the parD stability system of plasmid R1, is an inhibitor of DNA replication acting at the initiation of DNA synthesis.

The Kid and Kis proteins are the killer component and the antagonist belonging to parD, a killer stability system of plasmid R1. The Kid and Kis proteins have been purified, the second one as a C-LYT-Kis fusion that conserves the antagonistic activity of the Kis protein, but not its auto-regulatory potential. Kid inhibits in vitro replication of CoEl to a basal level without altering the superhelicity of the template but it does not substantially affect in vitro replication of P4, a DnaA, DnaB, DnaC and DnaG-independent replicon. Kid inhibits lytic induction of a lambda, prophage, but this inhibition can be neutralized by excess DnaB. In addition, a multicopy dnaB recombinant, but not a multicopy dnaG recombinant, prevents the toxicity associated with this protein. Inhibition of ColE1 replication by Kid in vitro is prevented by the C-LYT-Kis protein. Functional analysis indicates that the antagonistic activity of Kis is independent of its activity as a co-regulator of the parD promoter. It is also shown that C-LYT-Kis and Kid interact, forming a tight complex. These results strongly suggest that the toxicity of the kid protein is due to inhibition of DnaB-dependent DNA replication, and that direct protein-protein interactions are involved in the neutralization of the activity of the killer protein by the antagonist.

Bacterial Proteins↗
PubMedDate not supplied

Inhibition of the Synthesis of Proteins Needed for Epstein-Barr Virus Replication by Antisense RNA against the Zta Gene.

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(-), respectively. Synthesis of Zta protein was reduced in pZ(-)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(-)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication. Copyright 1997 S. Karger AG, Basel

Journal Article↗
PubMed1990

Restricted expression of herpes simplex virus lytic genes during establishment of latent infection by thymidine kinase-negative mutant viruses.

Infection of cells by herpes simplex virus (HSV) can lead to either lytic, productive infection or nonlytic, latent infection. The factors influencing this infection pathway decision are largely unknown. Thymidine kinase-negative mutant viruses can establish latent infection in neurons of mouse trigeminal ganglia but do not replicate productively in these cells. We show that during the early stages of establishment of latency by these mutants, expression of viral lytic genes is drastically reduced or undetectable as assayed by in situ hybridization. Thus, establishment of latent infection by HSV can occur despite severely restricted levels of lytic gene expression. This suggests that the block to productive replication during establishment of latent infection by HSV occurs before or early during the expression of alpha genes.

Animals↗
PubMed1980

Enhanced replication of herpes simplex virus type 1 in BHK-21 cells transformed by hamster sarcoma virus.

Untransformed and hamster sarcoma virus-transformed BHK-21 cells were compared for their ability to support productive infection with herpes simplex virus type 1. In both types of cells, infection with herpes simplex virus resulted in a lytic, productive cycle. However, inhibition of host cell RNA and DNA synthesis, transcription and replication of viral DNA, and production of infectious virus began earlier and reached higher levels in transformed cells.

Adsorption↗
PubMed1997

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Animals↗
PubMed1999

Expression of Epstein-Barr virus lytically related genes in African Burkitt's lymphoma: correlation with patient response to therapy.

A study on the Epstein-Barr virus (EBV)-associated malignancy (endemic) Burkitt's lymphoma (BL) was initiated on fine-needle-aspiration biopsies from 46 proven BL cases in Malawi. Gene expression that might correlate with patient serology (where high levels of antibodies to lytically related genes are commonly observed) was explored. In two-thirds of the cases, we identified the EBV BZLF1 replication activator intermediate early protein ZEBRA in varying quantities and to varying extents in cells by immuno-cytochemistry. The early lytic-cycle gene transcript BHLF1 was assessed positively by solid-phase hybridisation in over half of the same tumours. Evidence of transcription of these genes was confirmed on a smaller number of surgically removed fresh biopsies by RT-PCR. We asked whether our findings, which are generally counter to the established notion that EBV gene expression in BLs is restricted to the latent function, EBNA1, might offer some explanation for the differential responses to chemotherapy observed among African patients. Where the duration of follow-up was sufficient to assign the cases (37 in number) to one of 3 categories, namely, complete, partial or no response, a significant correlation between expression of the viral function ZEBRA and a positive patient response to treatment was found. Lack of this was associated with poor prognosis. Clinical data and EBV gene expression results support the postulate of subgroups of African BLs, the intermediate early antigen providing a marker of potential use in patient management.

Adolescent↗