Penetration

After the virus binds to a receptor, the next step toward successful infection is the introduction of the viral genome into the cytoplasm of the cell, In some cases, a subviral particle containing the viral nucleic acid is introduced into the cell, This particle may be the nucleocapsid of the virus or it may be an activated core particle, For other viruses, only the nucleic acid is introduced, The protein(s) that promotes entry may be the same as the protein(s) that binds to the receptor, or it may be a different protein in the virion,

For enveloped viruses, penetration into the cytoplasm involves the fusion of the envelope of the virus with a cellular membrane, which may be either the plasma membrane or an endosomal membrane, Fusion is promoted by a fusion domain that resides in one of the viral surface proteins, Activation of the fusion process is thought to require a change in the structure of the viral fusion protein that exposes the fusion domain, For viruses that fuse at the plasma membrane, interaction with the receptor appears to be sufficient to activate the fusion protein, In the case of viruses that fuse with endosomal membranes, the virus is first endocytosed into clathrin-coated vesicles and proceeds through the endosomal pathway, During transit, the clathrin coat is lost and the endosomes become progressively acidified, On exposure to a defined acidic pH (often ~5-6), activation of the fusion protein occurs and results in fusion of the viral envelope with that of the endosome,

A dramatic conformational rearrangement of the HA protein of influenza virus, a virus that fuses with internal membranes, has been observed by X-ray crystallography of HA following its exposure to low pH. HA, which is cleaved into two disulfide-bonded fragments HA1 and HA2, forms trimers that are present in a spike on the surface of the virion. The atomic structure of an HA monomer is illustrated in Fig. 1.5. HA1 (shown in blue) contains the domain (indicated with a star in the figure) that binds to sialic acid receptors, whereas HA2 (shown in red) has the fusion domain (yellow). The fusion domain, present at the N terminus of HA2, is hidden in a hydrophobic pocket within the spike near the lipid bilayer of the virus envelope. Exposure to low pH results in a dramatic rearrangement of HA that exposes the hydrophobic peptide and transports it more than 100 Â upward, where it is thought to insert into the cellular membrane and promote fusion. It is assumed that similar events occur for all enveloped viruses, whether fusion is at the cell surface or with an internal membrane.

After fusion of the viral envelope with a cellular membrane, the virus nucleocapsid is present in the cytoplasm of the cell. Virus entry by fusion can be very efficient. In some well-studied cases using cells in culture, almost all particles succeed in initiating infection.

For nonenveloped viruses, the mechanism by which the virus breaches the cell membrane is less clear. After binding

Cell Membrane Penetration

FIGURE 1.5 The folded structure of the influenza hemagglutinin and its rearrangement when exposed to low pH. (A) A schematic of the cleaved HA molecule. S is the signal peptide, TM is the membrane-spanning domain. HA1 is in blue, HA2 is in red, and the fusion peptide is shown in yellow. The same color scheme is used in (B) and (C). (B) X-ray crystallographic structure of the HA monomer. TM had been removed by proteolytic digestion prior to crystallization. The receptor binding pocket in HA1 is shown with a green star. In the virion HA occurs as a trimeric spike. (C) The HA2 monomer in the fusion active form. The fragment shown is produced by digesting with thermolysin, which removes most of HA1 and the fusion peptide of HA2. Certain residues are numbered to facilitate comparison of the two forms. The approximate location of the fusion peptide before thermolysin digestion is indicated with a yellow diamond. (B') Diagrammatic representation of the HA2 shown in (B), with a helices shown as cylinders and /} sheets as arrows. The disulfide link between HAl and HA2 is shown in ochre. The domains of HA2 are color coded from N terminus to C terminus with a rainbow. (C') Diagrammatic representation of the fusion-active form shown in (C). [Redrawn from Fields et al. (1996, p. 1361).]

FIGURE 1.5 The folded structure of the influenza hemagglutinin and its rearrangement when exposed to low pH. (A) A schematic of the cleaved HA molecule. S is the signal peptide, TM is the membrane-spanning domain. HA1 is in blue, HA2 is in red, and the fusion peptide is shown in yellow. The same color scheme is used in (B) and (C). (B) X-ray crystallographic structure of the HA monomer. TM had been removed by proteolytic digestion prior to crystallization. The receptor binding pocket in HA1 is shown with a green star. In the virion HA occurs as a trimeric spike. (C) The HA2 monomer in the fusion active form. The fragment shown is produced by digesting with thermolysin, which removes most of HA1 and the fusion peptide of HA2. Certain residues are numbered to facilitate comparison of the two forms. The approximate location of the fusion peptide before thermolysin digestion is indicated with a yellow diamond. (B') Diagrammatic representation of the HA2 shown in (B), with a helices shown as cylinders and /} sheets as arrows. The disulfide link between HAl and HA2 is shown in ochre. The domains of HA2 are color coded from N terminus to C terminus with a rainbow. (C') Diagrammatic representation of the fusion-active form shown in (C). [Redrawn from Fields et al. (1996, p. 1361).]

Overview to a receptor, somehow the virus or some subviral component ends up on the cytoplasmic side of a cellular membrane, the plasma membrane for some viruses or the membrane of an endosomal vesicle for others. It is believed that the interaction of the virus with a receptor, perhaps potentiated by the low pH in endosomes for those viruses that enter via the endosomal pathway, causes conforma-tional rearrangements in the proteins of the virus capsid that lead to the penetration of the membrane. In the case of poliovirus, it is known that interactions with receptors in vitro will lead to conformational rearrangements of the virion that result in the release of one of the virion proteins, called VP4. The N terminus of VP4 is myristylated and thus hydrophobic [myristic acid = CH3(CH2)12COOH]. It is proposed that the conformational changes induced by receptor binding result in the insertion of the myristic acid on VP4 into the cell membrane and the formation of a channel through which the RNA can enter the cell. It is presumed that other viruses also have hydrophobic domains that allow them to enter. A number of other viruses also have a structural protein with a myristilated N terminus that might promote entry. In some viruses, there is thought to be a hydrophobic fusion domain in a structural protein that provides this function. The entry process may be very efficient. The specific infectivity of reoviruses assayed in cultured cells can be almost one (all particles can initiate infection). In other cases, entry may be less efficient. The specific infectivity of poliovirus in cultured cells is usually less than 1%. Whether the specific infectivity of the virus is low when infecting humans is not clear, however.

Following initial penetration into the cytoplasm, further uncoating steps must often occur. It has been suggested that, at least in some cases, translation of the genomic RNA of plus-strand RNA viruses may promote its release from the nucleocapsid. In other words, the ribosomes may pull the RNA into the cytoplasm. In other cases, specific factors in the host cell, or the translation products of early viral transcripts, have been proposed to play a role in further uncoating.

It is interesting to note that bacteriophage face the problem of penetrating a rigid bacterial cell wall, rather than one of simply penetrating a plasma membrane or endosomal membrane. Many bacteriophage have evolved a tail by which they attach to the cell surface, drill a hole into the cell, and deliver the DNA into the bacterium. In some phage, the tail is contractile, leading to the analogy that the DNA is injected into the bacterium. Tailless phage are also known that introduce their DNA into the bacterium by other mechamisms.

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