Mechanisms of enveloped virus entry into animal cells

Abstract

The ability of viruses to transfer macromolecules between cells makes them attractive starting points for the design of biological delivery vehicles. Virus-based vectors and sub-viral systems are already finding biotechnological and medical applications for gene, peptide, vaccine and drug delivery. Progress has been made in understanding the cellular and molecular mechanisms underlying virus entry, particularly in identifying virus receptors. However, receptor binding is only a first step and we now have to understand how these molecules facilitate entry, how enveloped viruses fuse with cells or non-enveloped viruses penetrate the cell membrane, and what happens following penetration. Only through these detailed analyses will the full potential of viruses as vectors and delivery vehicles be realised. Here we discuss aspects of the entry mechanisms for several well-characterised viral systems. We do not attempt to provide a fully comprehensive review of virus entry but focus primarily on enveloped viruses.

Fig. 1

Attachment of virus particles to cell surface molecules. (A) Direct high affinity binding of a viral attachment protein (VAP) (red) to primary receptors (blue and green). The VAP depicted here has two receptor-binding sites, which allows it to attach to alternative receptors, expressed on different cell types. Examples: HIV-1 which can bind to both CD4 and galactosyl ceramide and SFV which can bind to MHC class I and an unknown receptor. (B) Adsorption of virus particles to the target cell surface by binding of the VAP to `pre-receptor' molecules (green), followed by high affinity binding to a primary receptor molecule (blue). Examples: recruitment of HSV by heparan sulphate and subsequent transfer to the tumour necrosis factor/nerve growth factor receptor homologue, and recruitment of adenovirus by an unknown receptor and transfer to the vitronectin receptor. (C) High affinity binding of a VAP to a receptor (green) induces conformational changes leading to the exposure of binding sites for a co-receptor (blue). Examples: primate lentiviruses (HIV-1, HIV-2 and SIV) which bind to CD4 and chemokine receptor co-receptors (NB: some tissue culture variants of HIV may be able to by-pass the CD4 binding step and interact directly with the chemokine receptor molecule (see Fig. 2; [62])

Fig. 2

Transition of VAP/fusion proteins to the fusogenic state after VAP binding. (A) Conformational changes in the VAP/fusion protein are triggered through interaction with the receptor. These changes induce fusion competent states of the fusion protein, as illustrated by the exposure of N-terminal fusion peptides (black). Examples: induction of avian leukaemia virus (ALV) fusion through binding to the LDLR-related protein. (B) Low pH-induced fusion: pH-dependent viruses (e.g., influenza virus, VSV and SFV) undergo receptor-mediated endocytosis after binding to cell surface receptors and fuse within intracellular organelles. These organelles are acidified by vacuolar H⁺ATPases. (C) Two step activation of a VAP/fusion protein. Interaction with a primary receptor exposes binding sites for a co-receptor. Upon binding of the co-receptor the VAP/fusion protein changes to the fusogenic state. Example: primate lentiviruses (HIV-1, -2 and SIV). (C′) In contrast to (C), some HIV Env proteins have a capacity to interact with the co-receptor (blue) alone without the requirement of the primary receptor. Binding to the primary receptor can still occur but is not obligatory.

Fig. 3

Comparison of the VAP/fusion proteins of HIV and influenza virus. The precursors of the HIV Env protein (top panel) and the influenza virus haemagglutonin (HA) (bottom panel) are aligned schematically to illustrate some hypothetical structure–functional similarities. The two precursor polypeptide chains are represented from their N-termini to the left to their C-termini to the right. The HIV Env encompasses approximately 860 residues and influenza HA 560. The C-termini of both molecules are cytoplasmic, or intravirional, but the cytoplasmic domain of HIV Env is considerably longer than that of HA (as represented by the segments to the right of the trans-membrane domains). During transport to the cell surface both precursors are cleaved as a prerequisite for subsequent fusion activity. The two resulting polypeptide chains of HIV Env, SU/gp120 (surface) and TM/gp41 (transmembrane), are non-covalently associated. Those of influenza virus, HA1 and HA2, are linked by a disulphide bond (intrachain disulphide bonds are not marked). SU and HA1 are responsible for the binding of the respective viruses to cell surface receptors, CD4 and sialic acid. Both receptor-binding sites are created by the juxtaposition in space of conserved residues that are non-contiguous in the amino-acid sequence. In SU of HIV these residues are situated in conserved regions on both sides of the V3 loop. The V3 loop influences the interactions with the chemokine-receptor co-receptors. The cleavage of the precursors creates novel N-termini in the transmembrane proteins. These N-terminal peptides share similar hydrophobic sequences and have been implicated in membrane fusion. C-terminal to the fusion peptides are potential `coil regions', two in the HIV TM extracellular domain (approx. residues 550–580 and 630–660 in the precursor), and one region in HA2 (residues 40–105). These regions have a propensity to form alpha helices and may form coiled-coils as a step in a series of events that activate the membrane-fusing capacities of the two proteins.