Viruses endocytosis

In addition to binding to sialic acid residues of the carbohydrate side chains of cellular proteins that the virus exploits as receptors, hemagglutinin has a second function in the infection of host cells. Viruses, bound to the plasma membrane via their membrane receptors, are taken into the cells by endocytosis. Proton pumps in the membrane of endocytic vesicles that now contain the bound viruses cause an accumulation of protons and a consequent lowering of the pH inside the vesicles. The acidic pH (below pH 6) allows hemagglutinin to fulfill its second role, namely, to act as a membrane fusogen by inducing the fusion of the viral envelope membrane with the membrane of the endosome. This expels the viral RNA into the cytoplasm, where it can begin to replicate. 

This fusogenic activity of influenza hemagglutinin is frequently exploited in the laboratory. If, for example, the virus is bound to cells at a temperature too low for endocytosis and then the pH of the external medium is lowered, the hemagglutinin causes direct fusion of the viral envelope with the plasma membrane infection is achieved without endocytosis. Similarly, artificial vesicles with hemagglutinin in their membrane and other molecules in their lumen can be caused to fuse with cells by first allowing the vesicles to bind to the plasma membrane via the hemagglutinin and then lowering the pH of the medium. In this way the contents of the vesicles are delivered to the recipient cell s cytoplasm. 

There is a dark side to receptor-mediated endocyto-sis in that viruses which cause such diseases as hepatitis (affecting liver cells), poliomyelitis (affecting motor neurons), and AIDS (affecting T cells) initiate their damage by this mechanism. Iron toxicity also begins with excessive uptake due to endocytosis. 

With animal cells, the whole virus particle penetrates the cell, being carried inside by endocytosis (phagocytosis or pinocytosis), an active cellular process. We describe some of these processes in detail later in this chapter. 

We might also note another important difference between animal and bacterial cells. Bacterial cells have rigid cell walls containing peptidoglycan and associated substances. Animal cells, on the other hand, lack cell walls. This difference is important for the way by which the virus genome enters and exits the cell. In bacteria, the protein coat of the virus remains on the outside of the cell and only the nucleic acid enters. In animal viruses, on the other hand, uptake of the virus often occurs by endocytosis (pinocytosis or phagocytosis), processes which are characteristic of animal cells, so that the whole virus particle enters the cell. The separation of animal virus genomes from their protein coats then occurs inside the cell. 

ErbacherP, Remy JS, Behr JP (1999) Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway. Gene Ther 6 138-145... 

Infection of CD4+ cells commences via interaction between gp 120 and the CD4 glycoprotein, which effectively acts as the viral receptor. Entry of the virus into the cell, which appears to require some additional cellular components, occurs via endocytosis and/or fusion of the viral and cellular membranes. The gp 41 transmembrane protein plays an essential role in this process. 

Some attempts have been made to rationally increase the efficiency of endosomal escape. One such avenue entails the incorporation of selected hydrophobic (viral) peptides into the gene delivery systems. Many viruses naturally enter animal cells via receptor-mediated endocytosis. These viruses have evolved efficient means of endosomal escape, usually relying upon membrane-disrupting peptides derived from the viral coat proteins. 

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. 

Living cells visualization of membranes, lipids, proteins, DNA, RNA, surface antigens, surface glycoconjugates membrane dynamics membrane permeability membrane potential intracellular pH cytoplasmic calcium, sodium, chloride, proton concentration redox state enzyme activities cell-cell and cell-virus interactions membrane fusion endocytosis viability, cell cycle cytotoxic activity... 

Binding to the cell surface proceeds at 0°C, but the cells are not infected (Helenius et al., 1980). When the cells are warmed to 37 C the virus is rapidly removed from the cell surface and infection ensues. In general there are two ways to envisage the entry of enveloped viruses into cells—either by penetration directly through the plasma membrane, or by endocytosis (engulfment by a plasma membrane-derived vesicle) (see Lonberg-Holm and Philipson, 1974). In both cases delivery of the nucleocapsid with the RNA would have to involve a fusion reaction between the viral envelope and either the cell surface membrane or the vesicle membrane. Paramyxoviruses are known to fuse their envelopes with the plasma membrane (see Hosaka and Shimizu, 1977). However, whether this process leads to productive infection has not yet been settled. 

Since their discovery in the early 1950s (14,15), caveolae had been considered to be uninteresting static organelles that have no capability for the uptake of particles. However, in the past two decades, caveolae have moved into the focus of many researchers because they seem to play an important role in the uptake of various agents, but this is not without controversy. In 2002, Thomsen et al. published strong evidence that caveolae are static fixed domains that are not involved in endocytosis (16), but it has also been reported that caveolae and caveolin can clearly be internalized—at least after specific stimuli, as shown with simian virus 40 (SV40) (17), or by treatment with okadaic acid (18). 

After attachment to the cell surface, the virus is located in clathrin-coated pits and is further internalized by an endocytosis similar to the receptor-mediated endocytosis of LDL (114,115). 

Pelkmans L, Kartenbeck J, Helenius A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 2001 3(5) 473 83. 

Marsh M, Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol 1980 142(3) 439 54. 

Sieczkarski SB, Whittaker GR. Differential requirements of rab5 and rab7 for endocytosis of influenza and other enveloped viruses. Traffic 2003 4(5) 333-343. 

Damm EM, Pelkmans L, Kartenbeck J, Mezzacasa A, Kurzchalia T, Helenius A. Clathrin- and caveolin-1-independent endocytosis entry of simian virus 40 into cells devoid of caveolae. J Cell Biol 2005 168(3) 477-488. 

Sieczkarski SB, Whittaker GR. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol 2002 76(20) 10455-10464. 

Infection begins when specific receptor sites on the virus recognize corresponding surface proteins on the host cell. The virus penetrates the host membrane by a mechanism resembling endocytosis and is encapsulated... 

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. 

Virus triggers endocytosis becomes enclosed in an endosome. 

Receptor-mediated endocytosis is exploited by some toxins and viruses to gain entry to cells. Influenza virus (see Fig. 11-24), diphtheria toxin, and cholera toxin all enter cells in this way. 

Viruses have many modes of life. They enter cells in various ways. Some enter through coated pits from which they are taken into lysosomes via endocytosis. Others are literally injected into the cells (See Box 7-C). Within cells some viruses are assembled in the nucleus, some in the cytoplasm, and some in membranes. The typical life cycle of a virus leads to rapid formation of large numbers of progeny. Within 20 minutes after entrance into a bacterial cell, a bacteriophage can... 

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. 

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