SEARCH · PubMed Health

Results for “Virus Internalization”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources

PubMed2006

N- and 6-O-sulfated heparan sulfates mediate internalization of coxsackievirus B3 variant PD into CHO-K1 cells.

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO(3)(6OSO(3))](n), but not basic fibroblast growth factor binding to [HexUA-GlcNSO(3)-HexUA-GlcNSO(3)-IdUA(2OSO(3))](n), was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO(3)(6OSO(3))](n) is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.

Animals↗

PubMed1994

The entry of Junin virus into Vero cells.

The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryocyte formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

Amantadine↗

PubMed1999

Incorporation of wild-type and C-terminally truncated human epidermal growth factor receptor into human immunodeficiency virus-like particles: insight into the processes governing glycoprotein incorporation into retroviral particles.

Previous results have indicated that incorporation of surface glycoprotein into retroviral particles is not a specific process and that many heterologous viral and cellular glycoproteins can be incorporated as long as they do not have long cytoplasmic C-terminal regions which were presumed to be sterically inhibitory. In this study, this concept has been directly examined by analyzing the incorporation of the wild-type human epidermal growth factor receptor (Wt-EGFR) and of a C-terminally truncated mutant of Wt-EGFR (Tr-EGFR) into human immunodeficiency virus (HIV)-like particles. Incorporation was directly analyzed at the protein level and by immunogold labelling of enriched HIV-like particles. In agreement with the above concept, Tr-EGFR, with only 7 C-terminal amino acids (aa), was efficiently incorporated into HIV-like particles. Incorporation of the Wt-EGFR species, with 542 C-terminal cytoplasmic aa, was reduced by a factor of about 5 in comparison to that of the Tr-EGFR species. However, the Wt-EGFR species was still very significantly present in the HIV-like particles. A series of control experiments verified that this represents genuine incorporation of Wt-EGFR into the membrane of HIV-like particles. These observations allow further speculation as to the processes governing glycoprotein incorporation into retroviral particles and indicate that the internal virus structure of HIV (in particular the matrix layer [MA]) can accommodate much larger heterologous cytoplasmic domains in incorporated glycoproteins than previously assumed.

Cell Line↗

PubMed2000

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.

Animals↗

PubMed2006

Suppression of coronavirus replication by inhibition of the MEK signaling pathway.

We previously demonstrated that infection of cultured cells with murine coronavirus mouse hepatitis virus (MHV) resulted in activation of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway (Y. Cai et al., Virology 355:152-163, 2006). Here we show that inhibition of the Raf/MEK/ERK signaling pathway by the MEK inhibitor UO126 significantly impaired MHV progeny production (a reduction of 95 to 99% in virus titer), which correlated with the phosphorylation status of ERK1/2. Moreover, knockdown of MEK1/2 and ERK1/2 by small interfering RNAs suppressed MHV replication. The inhibitory effect of UO126 on MHV production appeared to be a general phenomenon since the effect was consistently observed in all six different MHV strains and in three different cell types tested; it was likely exerted at the postentry steps of the virus life cycle because the virus titers were similarly inhibited from infected cells treated at 1 h prior to, during, or after infection. Furthermore, the treatment did not affect the virus entry, as revealed by the virus internalization assay. Metabolic labeling and reporter gene assays demonstrated that translation of cellular and viral mRNAs appeared unaffected by UO126 treatment. However, synthesis of viral genomic and subgenomic RNAs was severely suppressed by UO126 treatment, as demonstrated by a reduced incorporation of [3H]uridine and a decrease in chloramphenicol acetyltransferase (CAT) activity in a defective-interfering RNA-CAT reporter assay. These findings indicate that the Raf/MEK/ERK signaling pathway is involved in MHV RNA synthesis.

Animals↗

PubMed1999

Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes.

Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications.

Animals↗

PubMed1985

Nucleotide sequences responsible for generation of internally deleted Sendai virus defective interfering genomes.

The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a (Mr 1.24 x 10(6)) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5'-terminal genome and adjacent L gene sequences. DI RNA 7b (Mr, 0.70 x 10(6)) consisted of 100 3'-terminal nucleotides fused to 1900 5'-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c (Mr 0.55 x 10(6)) retained 420 3'-terminal and 1150 5'-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an MRNA encoding a fusion protein with a 14,000 Mr (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d (Mr 0.92 x 10(6)) retained 1027 3'-terminal nucleotides fused to 1600 bases from the 5'-terminus. It has an open reading frame for a 33,000 Mr N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication.

Base Sequence↗

PubMed1997

Resistance to infection with mouse hepatitis virus (MHV) in the cell clones derived from persistently infected DBT cells with the JHM strain of MHV.

PiD-10 and piD-11 cells that have been established from persistently infected DBT cells with the JHM strain of MHV (JHMV) were resistant to infection with JHMV. There was no significant difference in the amount of adsorbed virus among piD-10, piD-11 and DBT cells. When an expression of mRNA of the MHV receptor in piD-10 and piD-11 cells was analyzed by the RT-PCR method, no significant difference was observed in the intensities of the amplified products among piD-10, piD-11 and DBT cells. Treatment of virus-adsorbed cells with PEG, which induces fusion of the cellular membrane with the viral envelop, causes entry of virus particles into cells. There was no significant difference in the yields of virus between PEG-treated and PEG-untreated cells. The titers of infectious virus internalized into piD-10 and piD-11 cells were the same as those in DBT cells. When piD-10 and piD-11 cells were fused with PEG and infected with JHMV, the yields of infectious virion particles from the fused cells between piD-10 and piD-11 cells were significantly lower than those from the fused cells between DBT and piD-10 or piD-11 cells. The present study showed that resistance of piD-10 and piD-11 cells to JHMV infection is not due to an inhibition of JHMV entry into the cells.

Animals↗

PubMed2006

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

Cell Line, Tumor↗

PubMed1993

Effects of staurosporine on transcription by vesicular stomatitis virus.

The protein kinase inhibitor staurosporine has been found to inhibit vesicular stomatitis virus transcription in infected BHK cells. Both primary and secondary transcription were virtually abolished by 10 microM staurosporine. In contrast, transcription by purified virions, or by nucleocapsids isolated from them, was unaffected by staurosporine. Staurosporine inhibition did not occur at or prior to the uncoating step, since: (i) the drug was equally effective if added at t = 0 or at t = 1 hr after infection; (ii) immunofluorescence microscopy of infected cells in the presence of staurosporine showed that M protein was diffusely present in the cytoplasm, indicative of normal uncoating. Staurosporine caused a modest (< 25%) reduction in virus internalization from the cell surfaces. This was not sufficient to account for the transcription inhibition, however. Inhibition of transcription by staurosporine was not observed in extracts from infected cells, nor could it be induced by addition of uninfected cell cytosol to purified nucleocapsids. This suggests that inhibition of transcription in infected cells is secondary to substrate depletion or other cytotoxic effects of the drug. The addition of staurosporine to purified nucleocapsids, while not affecting cell-free transcription, inhibited phosphorylation of L protein completely and of P (NS) protein partially, providing addition evidence that at least two different kinases are present in nucleocapsids, and that the staurosporine inhibited ones are unnecessary for cell-free viral transcription.

Alkaloids↗

PubMed1997

Inhibition of tryptase TL2 from human T4+ lymphocytes and inhibition of HIV-1 replication in H9 cells by recombinant aprotinin and bikunin homologues.

The serine esterase TL2 from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL2 and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg15, Phe17, Glu52] aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL2 (> 80%). However, the [Leu15, Phe17, Glu52] aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL2 (68%). Our results show that the enzyme activity of purified tryptase TL2 is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL2 is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL2 is involved in the mechanism of virus internalization into human lymphocytes. The [Leu15, Phe17, Glu52] aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 micro M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-alpha-trypsin inhibitor. Only the single-headed variant [Arg94] delta 2 bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 micro M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.

Amino Acid Sequence↗

PubMed1999

pIRES-CD4t, a dicistronic expression vector for MACS- or FACS-based selection of transfected cells.

To facilitate the selection of cell lines expressing transfected genes of interest, a plasmid vector has been constructed that directs the co-expression of heterologous cDNAs and a 3'-positioned cassette encoding a truncated CD4 marker. An encephalomyocarditis virus internal ribosomal entry site (IRES) mediates translational initiation from this 3' cassette, and a cytomegalovirus promoter drives dicistronic transcript expression. To test the utility of this vector, a luciferase reporter gene was inserted, and this construct (pIRES-CD4t-luc) was electrotransfected into myeloid FDCW2 cells. As monitored by flow cytometry and luciferase assays, three rounds of magnetic cells sorting (MACS) yielded > or = 90% CD4t-positive cells with an average density of 17,000 CD4t molecules per cell. In ten clonal sublines analyzed, luciferase expression was uniformly high and stable over a test period of three months. Finally, a comparison of MACS- vs. FACS-based isolation of transfected cells showed two to three rapid rounds of MACS to be somewhat more effective. Thus, pIRES-CD4t should prove useful in the direct and rapid selection of relevant stably or transiently transfected cells.

Animals↗

PubMed1994

Analysis of the competition between nucleosome formation and transcription factor binding.

We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer binding factor (IBF) factor (which recent studies indicate is almost certainly cEBP beta). We find that histones and IBF are incapable of forming a ternary complex with a 159-base pair (bp) fragment of DNA containing a single IBF binding site and that histones and factor are mutually exclusive in binding. We have analyzed the various physical parameters of binding, in an attempt to understand how the factor might establish an exclusive binding in the cell. The stability of the nucleosome and the factor-DNA complex have been determined, and in addition a minimum value for the affinity of the histone octamer has been computed. We find that in simple competition the IBF can successfully compete, only if the substrate DNA is shorter than 140 bp. The relevance of these results is discussed in terms of a kinetic model for successful factor competition during the replication of the factor binding site in the cell.

Avian Sarcoma Viruses↗

PubMed1986

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.

Antibodies, Monoclonal↗

PubMed2005

Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.

Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.

Cell Line, Tumor↗

PubMed1998

A dicistronic retroviral vector and culture model for analysis of neuron-Schwann cell interactions.

A dicistronic retroviral gene delivery system and tissue culture model has been developed for studies of neuron-Schwann cell interactions at the single cell level. The dicistronic retroviral vector contains a multiple cloning site followed by the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES) and a green fluorescent protein gene. This design allows for 5'-cap dependent translation of any gene of interest and 5'-cap independent translation of green fluorescent protein (GFP) from a single dicistronic RNA. The culture model consists of dorsal root ganglia (DRG) explants grown in defined medium. Under these conditions the Schwann cell population is selectively expanded and infected by the retroviral vector, allowing for rapid transfer of genes of interest selectively to a large percentage of Schwann cells in coculture with neurons. Infected cells are subsequently identified in living cultures by their expression of GFP. Infected (GFP expressing) Schwann cells in contact with neurites continued to exhibit: (1) increased mitotic activity, (2) increased sensitivity to elevate intracellular calcium in response to extracellular application of ATP, and (3) myelination. This viral construct has the added advantage that it allows identification of cells expressing transgenes among a heterogeneous population by fluorescence microscopy, FACS, or flow cytometry.

Adenosine Triphosphate↗

PubMed1994

Binding of reovirus to receptor leads to conformational changes in viral capsid proteins that are reversible upon virus detachment.

A conformational change was detected in reovirus upon its attachment to mouse L fibroblasts. Specifically, the capsid proteins of cell-bound virions became more resistant to pepsin digestion. Similar observations were made using glutaraldehyde-fixed cells or plasma membranes instead of live cells, indicating that virus internalization was not necessary for this effect. This conformational change was totally reversible, since bound virions reverted back to the pepsin-sensitive state upon release from the cell surface. Not unexpectedly, a conformational change was also detected in the reovirus cell attachment protein sigma 1 when it alone bound to cells. The alteration was mapped, by deletion mutagenesis, to a region proximal to the N-terminal (virion-anchoring) end of the protein and was also found to be reversible. Structural changes in sigma 1 were also detectable following its interaction with sialic acid (conjugated to bovine serum albumin) shown previously to the minimal receptor determinant recognized by reovirus. These results suggest that upon cell attachment, a signal is transmitted from the C-terminal receptor-binding region of sigma 1 to the N terminus in a ripple-like progression that eventually leads to conformational changes in the other reovirus capsid proteins. An altered conformational state may be necessary for subsequent viral entry and programmed disassembly of viral capsids inside susceptible cells.

Animals↗

PubMed2000

Long-term transgene expression in mice infected with a herpes simplex virus type 1 mutant severely impaired for immediate-early gene expression.

The role of viral immediate-early (IE) gene expression in herpes simplex virus type 1 (HSV-1) latency was investigated. The HSV-1 multiple mutant in1312, defective for the expression of the virion transactivator VP16 and the IE proteins ICP0 and ICP4, was used as the parent for these studies. The coding sequences of the Escherichia coli lacZ gene, preceded by the encephalomyocarditis virus internal ribosome entry site, were inserted into the region of in1312 that encodes the latency-associated transcripts (LATs) such that transcription of the transgene was controlled by the LAT promoter. This insert has previously been shown to direct long-term latent-phase expression of beta-galactosidase in a wild-type HSV-1 genome (R. H. Lachmann and S. Efstathiou, J. Virol. 71, 3197-3207, 1997). The resulting recombinant, in1388, was apathogenic after inoculation into mice via the footpad and did not detectably replicate in dorsal root ganglia (DRG) or footpads. Mutant in1388 established latency in DRG, and beta-galactosidase was expressed in increasing numbers of neurons over the first 25 days of infection. During latency, more than 1% of neurons in ganglia that innervate the footpad expressed beta-galactosidase, with the number of positive cells remaining constant for at least 5 months. Rescue of the VP16, ICP0, or ICP4 mutations of in1388 did not affect the number of beta-galactosidase-expressing neurons detected during latency. The results demonstrate that HSV-1 mutants severely impaired for IE gene expression are capable of establishing latency and efficiently expressing a foreign gene product under control of the LAT promoter.

Animals↗