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PubMed2026

Rab9 depletion enhances human adenovirus type 26 transduction efficiency through increased internalization and reduced late endosomal/lysosomal retention.

Understanding intracellular trafficking is central to decoding viral pathogenesis and engineering optimized viral vectors. How a virus or vector is routed through the endocytic pathway directly dictates its genome release, immune sensing, and overall transduction efficiency. Human adenovirus type 26 (HAdV-D26) presents a promising platform for vector design due to its low preexisting immunity, potent immune stimulation, scalable production, and versatile genetic engineering capacity. Although increasingly significant, the fundamental mechanisms governing HAdV-D26 intracellular trafficking are still not fully understood. Our study demonstrates that compared to well-described human adenovirus type 5 (HAdV-C5), HAdV-D26 undergoes prolonged intracellular trafficking, transiently localizing to early endosomes before residing in late endosomes/lysosomes for up to four hours post-infection. Inhibition of lysosomal acidification modestly enhances HAdV-D26 transduction efficiency, whereas blocking transport from early to late endosomes/lysosomes does not. Strikingly, Rab9 knockdown reduces HAdV-D26 late endosomal/lysosomal localization while increasing both virus internalization and genome delivery to the host cell nucleus. These findings indicate that late endosomal sorting pathways actively influence HAdV-D26 infection outcomes. By identifying a previously unappreciated role for Rab9 in adenovirus transduction, our results provide new mechanistic insight into HAdV-D26 intracellular trafficking, highlight serotype-specific differences in adenovirus entry pathways, and identify endosomal trafficking steps that may be targeted to improve adenoviral vector performance.

Humans↗
PubMed2000

Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells.

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.

Adenoviruses, Human↗
PubMed2006

Calcium-dependent viral internalization is required for adenovirus type 7 induction of IL-8 protein.

The host response to adenovirus (Ad) infection involves induction of cytokines in lung epithelia. We have demonstrated induction of the lung neutrophil chemokine interleukin-8 (IL-8) by Ad7, a major lung pathogen, in A549 lung epithelial cells and lung tissue through activation of the Erk signaling pathway. However, the mechanism of IL-8 induction is still unclear. In this paper, we first showed that Ad7 viral gene expression is not essential for IL-8 induction as psoralen-UV inactivation of Ad7 did not affect IL-8 mRNA induction or IL-8 protein induction in A549 cells. We then inhibited internalization of Ad7 by treatment of A549 cells with EGTA in calcium-free medium during exposure to Ad7. We verified that this treatment inhibited Ad internalization by confocal microscopy, FACS analysis and Ad E1A and fiber mRNA expression. Preventing internalization by calcium depletion did not inhibit Erk activation by Ad7. However, calcium-dependent internalization was required for IL-8 protein production in Ad7 exposed cells. This is not likely due to an effect of calcium depletion on downstream Erk signaling or IL-8 protein production since calcium depletion did not block IL-8 protein production stimulated by PMA, and because addition of EGTA subsequent to Ad7 internalization also did not prevent Ad induction of IL-8. These studies indicate that Ad7 internalization is calcium-dependent and is required for IL-8 protein induction upon Ad7 infection. Ad7 induction of Erk is independent of calcium and does not require virus internalization.

Adenoviruses, Human↗
PubMed1981

Aberrant glycoprotein mRNA synthesized by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesized an aberrant polyadenylated mRNA in vivo and in vitro. No normal glycoprotein message could be detected among the in vivo transcription products. The abnormal RNA contained a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message. The polyadenylate is located at the 3' end of the molecule and is most probably encoded by the remnant polymerase gene polyadenylation signal. This aberrant RNA may be synthesized because of either a failure to terminate transcription at the end of the glycoprotein gene or an inability to process an abnormal polycistronic precursor.

Animals↗
PubMed1983

Conditional synthesis of an aberrant glycoprotein mRNA by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesizes an aberrant polyadenylated mRNA (G*) containing a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message (R. C. Herman and R. A. Lazzarini, J. Virol. 40:78-86, 1981). The heat-resistant polymerase appears to play a role in the synthesis of the abnormal G* RNA. The synthesis of G* correlated directly with the presence of the heat-resistant L protein on the defective interfering particle template. Chimeric defective interfering particles produced by passaging DI-LT with a helper virus that encodes the wild-type vesicular stomatitis virus polymerase did not synthesize G*. The subsequent passage of the chimeric DI-LT with a heat-resistant helper virus restored the ability to synthesize the G* transcript. These results imply that the regulatory signals normally present at the vesicular stomatitis virus G/L intercistronic boundary may be preserved in DI-LT. These sequences are only conditionally functional because they are recognized correctly by the wild-type but not by the heat-resistant polymerase.

Defective Viruses↗
PubMed2000

Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.

Animals↗
PubMed1988

Early events of importance in determining host cell permissiveness to mouse hepatitis virus infection.

Three categories of cell lines are described which differ with respect to their permissiveness to mouse hepatitis virus (MHV), strain A59. Fully permissive L-2 cells gave rise to 100- to 1000-fold higher numbers of infectious centres than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, semi-permissive cells (LM, LM-K or C-1300) were as efficient in replicating viral RNA, protein and progeny virions as fully permissive L-2 cells. This result suggested that LM, LM-K and C-1300 cells were deficient in their ability to permit full expression (as compared to L-2 cells) of an early event in MHV infection. Assays of radiolabelled MHV binding to cells of all three categories (L-2, LM, LM-K and C-6) and of infectious MHV binding to L-2 and LM-K cells showed no correlation between virion binding and degree of permissiveness to MHV infection. Internalization of MHV virions into L-2 and LM-K cells, as assayed by proteinase K-resistant infectious centres, showed that, in both cases, maximum virion uptake was complete by approximately 40 min post-inoculation. Direct assays of infectious virion uptake showed similar numbers of internalized viruses (only a threefold difference between L-2 and LM-K cells, as compared to a 500-fold difference in infectious centres). Attempts to enhance MHV uptake into LM-K cells relative to L-2 cells, with DEAE-dextran or the cytoskeleton-disrupting drugs colchicine and cytochalasin B, were unsuccessful, further suggesting that the ability of LM-K cells to internalize the virus was not lacking. The results suggest that MHV infection of at least some semi-permissive cells, such as the LM-K line, is limited by a process which chronologically correlates with virion uncoating. Since LM-K cells have been shown previously to be resistant to membrane fusion in MHV infection, it is postulated that they may also restrict uncoating of MHV by limiting the degree of normal endosomal membrane fusion with the viral envelope.

Animals↗
PubMed2006

Application of bovine viral diarrhoea virus as an internal control in nucleic acid amplification tests for hepatitis C virus RNA in plasma-derived products.

Plasma-derived products are produced from plasma via fractionation and chromatography techniques, but can also be produced by other methods. In the performance of nucleic acid amplification tests (NAT) with plasma-derived products, it is necessary to include an internal control for the monitoring of all procedures. In order to avoid false negative results, we confirmed the usefulness of the bovine viral diarrhoea virus (BVDV) for use as an internal control in the detection of hepatitis C virus (HCV) RNA in plasma-derived products. These products, which were spiked with BVDV, were extracted and then NAT was performed. Specificity and sensitivity were determined via the adjustment of primer concentrations and annealing temperatures. BVDV detection allows for validation in the extraction, reverse transcription, and amplification techniques used for HCV detection in plasma-derived products.

Animals↗
PubMed1983

Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis.

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 min to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase-catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.

Animals↗
PubMed2006

Vaccinia virus interactions with the cell membrane studied by new chromatic vesicle and cell sensor assays.

The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.

Animals↗
PubMed1982

Evidence for two T-helper populations with distinct specificity in the humoral response to influenza A viruses.

Virus specificity of T-helper cells for the humoral antibody response to influenza A viruses was studied with a hapten-carrier secondary adoptive transfer system, using whole virus, or viral components inserted into liposomes as carrier with B cells primed to DNP human gamma globulin. Evidence was obtained for two distinct T-helper cell populations from mice primed by influenza infection: a T-helper cell cross-reactive for all type A influenza viruses and a second T-helper population specific for the variant haemagglutinin. In vivo the virus cross-reactive T helpers recognized whole virus, but did not recognize isolated surface glycoproteins or internal virus proteins.

Animals↗
PubMed2002

Vitamin B12 stalls the 80 S ribosomal complex on the hepatitis C internal ribosome entry site.

The effect of cyanocobalamin (CNCbl, vitamin B12) on hepatitis C virus internal ribosome entry site (HCV IRES)-dependent initiation of translation was studied by ribosomal toeprinting and sucrose gradient centrifugation analysis. These results suggested that CNCbl did not inhibit HCV IRES-dependent translation by a competitive binding mechanism. CNCbl allowed 80 S elongation complex formation on the mRNA, but stalled the initiation at that point, effectively trapping the 80 S ribosomal complexes on the HCV IRES. CNCbl had no effect on cap-dependent mRNA, consistent with the known mRNA specificity of this translational inhibitor. To help elucidate the mechanism, comparative data were collected for the well-characterised translation inhibitors cycloheximide and 5'-guanylyl-imidophosphate. Although CNCbl stalled HCV IRES-dependent translation at approximately the same step in initiation as cycloheximide, the mechanisms of these two inhibitors are distinct.

Base Sequence↗
PubMed2001

Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER.

Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.

Animals↗
PubMed1989

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.

Animals↗