The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.
Adenoviridae ; physiology ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Cytoplasm ; virology ; Endocytosis ; Endosomes ; virology ; Genetic Vectors ; Humans ; Microtubules ; Virus Internalization

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Humans ; Autophagy ; Cell Death ; Cell Nucleus ; Human Umbilical Vein Endothelial Cells/virology* ; Dengue ; Proteome

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining, DNA ladder, Caspase 3 activity and flow cytometry, etc. The results showed that nuclear staining of infected cells with DAPI showed gradually morphological changes of the nuclei. As shown in the paper, a canonic oligonucleosome-sized DNA ladder was observed in cells harvested at 24h, 48h and 72h post-infection, confirming that DNA fragmentation was induced by RHDV infection. The results of flow cytometry showed that about 63% of cells were in apoptosis at 48h post-infection. Besides, we also demonstrated that the activation of Caspase 3 occurred during the infection process. In conclusion, our results showed that apoptosis in RHD might be determinant in the development of the pathogenesis of RHD.
Animals ; Apoptosis ; Caliciviridae Infections ; genetics ; physiopathology ; veterinary ; virology ; Caspase 3 ; metabolism ; Cell Line ; Cell Nucleus ; genetics ; virology ; DNA Fragmentation ; Hemorrhagic Disease Virus, Rabbit ; physiology ; Rabbits

Subcellular localizaton of HBcAg have been found to be related to the activity of liver disease and HBV replication. The aim of this study was to determine whether the degree of expression of HBcAg in the hepatocyte nucleus and cytoplasm reflects the level of viral replication and histological activity in chronic HBV infection. A total of 102 patients with biopsy proven chronic hepatitis B were included. There was a highly significant correlation between the levels of HBV DNA in serum and the degree of expression of HBcAg in the nucleus for HBeAg-positive(p=0.000) and negative patients(p=0.04). There was a highly significant, correlation between the degrees of expression of HBcAg in hepatocyte cytoplasm and histologic activities (p<0.01) for HBeAg-positive patients. The degrees of expression of HBcAg in the hepatocyte cytoplasm correlated positively with the lobular activities (p<0.01), but not correlated with the portal activity and fibrosis for HBeAg-negative patients. In conclusion, in the young patients with chronic B viral hepatitis, the degree of expression of HBcAg in the hepatocyte nucleus may affect viral load, and the degree of expression of HBcAg in the hepatocyte cytoplasm may affect histologic activities of liver disease.
Male ; Liver/pathology/virology ; Humans ; Hepatocytes/pathology/*virology ; Hepatitis B, Chronic/*pathology/*virology ; Hepatitis B e Antigens/metabolism ; Hepatitis B Core Antigens/*metabolism ; DNA, Viral/blood ; Cytoplasm/virology ; Cell Nucleus/virology ; Adult ; Adolescent

This study was performed to investigate the effects of different regions of the Autographa califor nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localization in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect Sf9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluorescence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain (2-45aa) of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells; and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25-EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot-like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the cells transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well; and the sequence downstream of the N-terminal domain also affects trafficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most cases, suggesting that the E25 likely exists and functions as dimmers or polymers. Production of infectious BV was dramatically reduced and even completely eliminated in most cases, either in the absence or presence of the native e25.
Amino Acid Motifs ; Animals ; Cell Nucleus ; metabolism ; virology ; Mutation ; Nucleopolyhedrovirus ; chemistry ; genetics ; physiology ; Protein Transport ; Spodoptera ; virology ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virus Release ; Virus Replication

Cell apoptosis induced by duck reovirus (DRV)in duck embryo fibroblasts (DEF) was ascertained by light microscope and electron microscopy, DNA Ladder, flow cytometry and fluorescent microscopy. Typical morphological apoptotic features including cell shrinkage and condensation, margination of nuclear chromatin were observed under light microscope and the formation of apoptotic bodies by electron microscopy. DNA ladder was shown by DNA fragment analysis at 24-144h post infection. Flow cytometry showed that the cell apoptosis appeared at 24h and reached it's crest-time at 72-96h, decreased at 144h. Fluorescent microscopy showed that the apoptotic cells which showed green fluorescence appeared at 24h, the number of dead cells which showed red fluorescence increased with the time went by. The results above confirmed that the apoptosis of DEF was successfully induced by DRV.
Animals ; Apoptosis ; physiology ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; DNA Fragmentation ; Ducks ; Embryo, Nonmammalian ; cytology ; Fibroblasts ; cytology ; ultrastructure ; virology ; Flow Cytometry ; Host-Pathogen Interactions ; Microscopy, Electron, Transmission ; Reoviridae ; physiology

Nuclear actin which plays a key role in many nucleic processes has become a research hotspot. Baculovirus is the only reported pathogen using nuclear actin to replicate and proliferate. However, little is known about the mechanism of monomeric G-actin accumulation within nuclei of baculovirus-infected cells. It has been reported that AcMNPV ie-1, pe38, ac4, he65, ac102, and ac152 could be required for mediating nuclear localization of G-actin from transiently transfected results in TN-368 cells. In this paper, we found that IE1, AC152, PE38, AC102 localized in the whole cell and PE38, AC102 localized in the nuclear mainly, while both AC4 and HE65 localized in cytoplasm and could be mediated into the nucleus by AC102 and IE1 respectively for the first time. And ie-1 or pe38, ac4, he65 could mediate nuclear G-actin to accumulate partly, while these four genes were sufficient for recruiting G-actin accumulation within the nucleus when driven by promoter OpIE2. Determining the functions of each of these AcMNPV NLA gene products will advance our understanding of baculovirus biology and function of nuclear actin.
Actins ; genetics ; metabolism ; Animals ; Cell Nucleus ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Nucleopolyhedrovirus ; genetics ; metabolism ; Promoter Regions, Genetic ; Protein Transport ; Spodoptera ; metabolism ; virology ; Viral Proteins ; genetics ; metabolism

Bel1, a transactivator of prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have shown that Bel1 bears a nuclear localization signal (NLS), but its amino acid sequence remains unclear and the corresponding importins have not been identified. In this report, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment could direct nuclear localization, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. Point mutation experiments revealed that K218, R219, and R221 are essential for the nuclear localization of Bel1. The results of the GST-pulldown showed that the Bel1 fragment with residues 215-223, which bears the NLS, interacts with KPNA1, KPNA6, and KPNA7. This result suggests that KPNA1, KPNA6, and KPNA7 maybe involved in Bel1 nuclear translocation.
Cell Line ; Cell Nucleus ; genetics ; metabolism ; virology ; Humans ; Nuclear Localization Signals ; genetics ; metabolism ; Protein Binding ; Protein Transport ; Retroviridae Infections ; genetics ; metabolism ; virology ; Retroviridae Proteins ; chemistry ; genetics ; metabolism ; Spumavirus ; chemistry ; genetics ; physiology ; Trans-Activators ; chemistry ; genetics ; metabolism ; alpha Karyopherins ; genetics ; metabolism

Tegument protein VP22 is encoded by Pseudorabies Virus (PRV) UL49. To identify the nuclear localization signals of UL49, it is necessary to determine the transport mechanism and biological functions of the VP22 protein. In this study, we identified two nuclear localization signals from UL49, NLS1 (5RKTRVA ADETASGARRR21) and NLS2 (241PGRKGKV247). The functional nuclear localization signal (NLS) of UL49 was identified by constructing truncated or site-specific UL49 mutants. The deletion of both NLS1 and NLS2 abrogated UL49 nuclear accumulation, whereas the deletion of NLS1 or NLS2 did not. Therefore, both NLS1 and NLS2 are critical for the nuclear localization of UL49. And our resuts showed that NLS2 is more important in this regard.
Animals ; COS Cells ; Cell Nucleus ; metabolism ; virology ; Cercopithecus aethiops ; Herpesvirus 1, Suid ; chemistry ; genetics ; metabolism ; Humans ; Nuclear Localization Signals ; Protein Transport ; Pseudorabies ; metabolism ; virology ; Viral Structural Proteins ; chemistry ; genetics ; metabolism