Endocytosis, viral particles

Viruses are complex particles, entering the cells by fusion of their envelope to the plasma membrane or by endocytosis followed by the escape of the capsid by membrane fusion or lysis (Sodeik, 2000). The diameter of the viral particle could be several hundred nanometers, implying a very inefficient diffusional movement in the cytoplasm, based on those physicochemical considerations that were discussed above (Kasamatsu and Nakanishi, 1998). Despite these limitations, those viruses that replicate in the nucleus have evolved sophisticated mechanisms to ensure a highly efficient nuclear delivery of their genetic material. Since these mechanisms may provide a conceptual framework to design novel non-viral delivery systems, we shall review some of the key elements that account for the nuclear targeting of certain viruses. 

Adenovirus vectors are known to be taken into cells by endocytosis and to be released from endosomes by a well regulated process, assumed to be highly efficient [84]. It is therefore somewhat surprising that PCI is able to increase the number of adenoviral transduced cells by up to 30-fold. Nevertheless, PCI with adenoviral vectors has been tested in several different cell lines, and in all cases improved transduction has been observed [85]. The adenovirus activated by means of PCI seems to follow the same cellular pathways as for conventional adenovims infection, i.e., the fraction of transduced cells followed a linear relationship with the Coxsackie and Adenoviral Receptor (CAR) level of the cells and is integrin dependent. Furthermore, PCI increase the number of nuclearly located viral DNA molecules as measured by real-time PCR and fluorescence in situ hybridization (FISH) [86]. The results so far indicate that the main cause of the PCI effect on transduction with adenovirus is related to enhanced release of the viral particles from the endocytic vesicles into the cytosol. In accordance with what has been found for PCI of plasmids the adenovirus may be delivered after the photochemical treatment (unpublished results). However, adenovirus may be delivered up to 12 h after the photochemical treatment, which is longer than what is effective for PCI of plasmids [43, 87]. 

After the binding, the virus may use various means of penetration (a) direct penetration, via the translocation of the entire virus through the cytoplasmic membrane (b) endocytosis, which is mediated by receptors, resulting in the formation of intercytoplasmic vesicles containing many viral particles (c) direct fusion of the viral envelope with the cytoplasmic membrane. 

Non-enveloped viruses generally use the first two penetration mechanisms, while the enveloped viruses enter a cell by endocytosis followed by binding with the membrane of an endosome. In addition to this mechanism, the enveloped viruses fuse directly with the cell membrane. The fusion of the viral envelope with the cell membrane requires the interaction of the glycoproteins of the virus with a cell receptor. After the internalization of the viral particle, the genome is freed for later expression. This process is known as unwrapping and it involves both cellular and viral enzymes. 

Once recognition and subsequent tight binding have occurred, the virus must penetrate the cell membrane of the host cell. Those viruses that have an outer envelope become engulfed by the cell membrane in a process called pinocytosis. The viral envelope becomes fused with the cell membrane and ultimately this breaks open to release the virion into the cytoplasm of the host cell. Its coat protein is then digested and the genome (DNA or RNA) is revealed. Viruses that do not possess an outer envelope also associate with the host cell membrane but in this case a pore opens up and the viral particle passes through it into the cytoplasm (endocytosis), where it is uncoated. 

Mechanisms Amantadine and rimantadine inhibit the first steps in replication of the influenza A and rubella viruses (Figure 49-1). These steps involve viral adsorption to the host cell membrane, penetration into the cell via endocytosis. and viral particle uncapping. The inhibitory action of these drugs may be due to their alkaline reaction, which raises the endo-somal pH. At low concentrations, amantadine also binds to a specific protein in the surface coat of the influenza virus to prevent fusion. Drug-resistant influenza A virus mutants can emerge and infect contacts of patients in treatment. 

The initial step in the sequence of events leading to influenza virus infections in mammalian hosts is mediated by the multiple attachment of virus particles to host sialoside receptors in the nasopharynx [41]. These receptors consist largely of cell surface sialylated glycoproteins and gangliosides. The subsequent steps involve receptor-mediated endocytosis with ensuing release of the viral nucleo-plasmid. The first event responsible for the receptor-virus interaction is therefore an attractive target for both antiviral and related microbial intervention. 

Budding and release of progeny virus. B. Replicative cycle of an influenza virus, an example of an RNA virus. 1. Attachment. 2. Endocytosis. 3. Influx of H+ through M2 protein. 4. Fusion of the viral envelope with the endosomes membrane, dissociation of the RNP complex, and entry of viral RNA into the nucleus. 5. Synthesis of viral mRNA by viral RNA polymerase. 6. Translation of viral mRNA by host cell s ribosomes. 7. Replication of viral RNA, using viral RNA polymerase, via cRNA replicative form. 8. Assembly of virus particles, and 9. Budding and release of progeny virus. 

This is in contrast to viruses, where the virus particles also show active transport when present in the cytosol after fusion with the plasma membrane or endosomal membrane [60-62], This is due to the ability of specific proteins of the virus particle to bind motor proteins. Single-particle tracking reveals that the quantitative intracellular transport properties of internalized non-viral gene vectors (e.g., polyplexes) are similar to that of viral vectors (e.g., adenovirus) [63]. Suk et al. showed that over 80% of polyplexes and adenoviruses in neurons are subdiffusive and 11-13% are actively transported. However, their trafficking pathways are substantially different. Polyplexes colocalized with endosomal compartments whereas adenovirus particles quickly escaped endosomes after endocytosis. Nevertheless, both exploit the intracellular transport machinery to be actively transported. 

Caveolae-mediated endocytosis is involved in viral transfection. This route can therefore be used for the delivery of oncolytic genetic materials by a viral vector [120]. Macropinocytosis is a relatively non-specific process which allows uptake of large particles up to the micron size range [121]. It is likely useful for the delivery of systems like solid lipid nanoparticles and PLN, which are... 

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