Retrovirus Attachment and Entry | Overview of Retrovirus Infection
Retroviruses are distinct from other types of viruses due to their unique flow of genetic information. Once a retrovirus has gained entry into a host cell, the genetic material is converted via reverse transcription from RNA to DNA, a process that is essentially backwards from normal transcription in which a cell converts DNA into RNA. This "backwards" flow of genetic information is where retroviruses derive their name.
Retroviruses are important tools for gene therapy because of their ability to integrate into the host cells genome and obtain long term gene expression. In fact, retroviruses are currently the second leading vector of choice in clinical trails. However, to utilize retroviral vector for gene delivery in humans it must be targeted to deliver its payload to a specific tissue or cell type. In order to develop effective targeting strategies it is imperative to understand the mechanism by which retroviral virions bind and enter cells.
The envelope glycoprotein complexes of retroviruses are expressed as a trimer of two polypeptides, an external glycosylated hydrophilic surface domain (SU) and a membrane-spanning transmembrane domain (TM). The SU portion of the viral glycoprotein harbors the determinants of the specificity of the virus for its cognate cellular receptor and therefore is vital to viral tropism. The TM subunit functions to anchor the complex into the viral envelope and is critical for fusion between cellular and viral membranes. Together they form a knob or knobbed spike on the surface of the virus and both are required for viral entry. Both the SU and TM domains are encoded by the viral env gene and are translated from spliced mRNA as a single polyprotein precursor that must be proteolytically cleaved by host proteases during transport to the cell surface. Cleavage of the polyprotein precursor occurs in the Golgi by the cellular protease furin. After cleavage, the products of the env gene are transported in vesicles to the plasma membrane where they are incorporated onto budding virions.
The SU portion of the glycoprotein is entirely located on the outside of the viral membrane. However, it remains attached via covalent or non-covalent interactions with the TM domain. The SU subunit contains domains responsible for the interaction with cell surface receptors including the receptor binding domain (RBD). It also contains a variable length N-terminal signal sequence (SP) that is recognized by signal recognition particle (SRP) and used to direct the precursor to the rough endoplasmic reticulum where it can undergo glycosylation, folding, oligomerization, and transport to the Golgi apparatus. Sequence comparisons of the SU domains from murine type C retroviruses have illustrated three highly variable regions; variable region A (VRA), variable region B (VRB), and the proline-rich region (PRR). VRA is found about 50 amino acids from the amino terminus of the SU domain and contains two conserved cysteine residues. VRB is found about 40 amino acids downstream of VRA and contains one conserved cysteine residue. The PPR domain is found downstream of VRB and is approximately 50 amino acids long. Studies have shown that the first two variable regions (VRA and VRB) determine the receptor specificity of the ecotropic Fr-MLV gp70. Evidence also suggests that the VRA region of Mo-MLV recognizes the receptor, while downstream regions such as VRB and proline-rich regions are important for stabilization of the receptor-specific structure on the surface of the virion. These regions confer specificity to different receptors and this specific interaction is the major determinant of the tropism of retroviruses as cells lacking the receptor are nonpermissive for viral entry.
The TM subunit is anchored in the cellular membrane and includes an extracellular fusion domain (FP), a membrane-spanning segment (tmd), a cytoplasmic tail (cty), and the R peptide (R); a 16 amino acid sequence at the C-terminal end that is cleaved off by the viral protease (PR) after the virion has been released from the cell. Studies have indicated that this sequence functions to inhibit the fusogenic activity of the glycoprotein complex in infected cells until they have been incorporated into a mature virion. Subsequent cleavage is thought to activate the fusogenic activity of the virus.
Retrovirus infection is initiated by the binding of the surface (SU) portion of the viral envelope glycoprotein to specific cellular receptors expressed on the cell surface. In the case of ecotropic MLV, the process is initiated by interaction with the 622 amino acid transmembrane receptor protein mouse cationic amino acid transporter (mCAT-1) located on the cell surface. The ecotropic virus can not infect human cells, presumably because the sequence of CAT-1 is divergent between murine and human receptors. The transporter has 14 membrane-spanning domains and identification of the virus binding region has been determined by exchanging portions of the genes that encode the permissive mouse and nonpermissive human molecules. These studies have shown that the third extracellular loop of the transporter binds to the SU domain of the virus, allowing viral attachment. In contrast, the amphotropic virus can infect both murine and non-murine cells by binding to the rat phosphate transporter Ram-1 or its human homologue Pit-2. The sequence of this receptor is highly conserved across species, thereby allowing infection of multiple species. Binding of either virus to its receptor (mCAT-1 or Pit-1) is thought to trigger a conformational change in the TM subunit of the glycoprotein, which in turn exposes the protein’s hydrophobic fusion peptide allowing mixing of viral and cellular membranes and subsequent entry of the viral capsid into the host cell cytoplasm. Displayed glycoproteins are biologically active and upon recognition of cognate cellular receptors undergo dramatic conformational rearrangement releasing energy thought to drive fusion; although at present this process is incompletely understood.
Enveloped viruses enter cells by fusion of the viral membrane with the plasma membrane of the target cell. However, fusion of two membrane bilayers is not a spontaneous process. There is a large kinetic barrier that can be reduced by viral fusion proteins which utilize the free energy liberated during a protein conformational change and act as a catalyst to fusion by pulling membranes together. For most retroviruses, a conformational change in the fusion protein can be induced by binding of the viral glycoprotein to a ligand (cellular receptor). However, in the case of Avian leukosis virus (ALV), fusion is induced in response to the increasing concentration of protons found within the endosome. Upon conformational change, the released energy is used to drive the fusion protein into the host cellular membrane, forming a bridge complex between heterologous membranes, via the viral membrane anchor in the C-terminal transmembrane domain. Next, the extended trimeric conformation, bridging the viral and target membranes, drives membrane merger by folding back on itself and forming a hairpin sturcture. This distortion lowers the energy barrier leading to the formation of a hemifusion stalk. Protein refolding then allows the formation of a pore and fusion of the two adjacent membranes. This process allows viral entry into the host cell cytoplasm.
It is generally acknowledged that there are two distinct pathways by which the interactions between viral glycoproteins and cellular receptors mediate membrane fusion, pH dependent and pH independent. Several enveloped viruses such as influenza and VSV require the increasingly acidic compartments of the endosome to initiate fusion. Influenza attaches to host cells via interactions between sialic acid containing glycoproteins or glycolipids and the viral hemagglutinin glycoprotein. It is internalized via both clathrin-dependent and clathrin-independent receptor-mediated endocytosis. Viruses utilizing the clathrin-dependent pathway enter cells in clathrin-coated pits (CCPs) and are trafficked from early to late endosomes where exposure to an increasing concentration of protons causes conformational changes within the viral hemagglutinin protein repositioning the fusogenic peptide into a conformation that allows interaction with the endosomal membrane and fusion with the viral envelope as previously discussed.
For the influenza viruses, interactions between the viral hemagglutinin glycoprotein and the cellular receptor are not sufficient to mediate fusion. Transport via endocytosis to the acidic environment of the endosome is required to fully activate the viral hemagglutinins fusogenic activity. In contrast, the majority of retroviruses are not dependent on exposure to an acidic environment but rather interactions between the viral glycoprotein and a cognate cellular receptor provide a conformational change that is adequate to activate the fusogenic activity of the complex, which results in merger of the viral and cellular membranes into one continuous membrane allowing viral entry into the host cell cytoplasm. These two distinct pathways suggest both differences and similarities exist between retrovirus and influenza virus membrane fusion.
The development of viable targeting strategies has been made possible by exhaustive scientific research on several retroviral Env glycoproteins that examined their ability to tolerate amino acid insertions, deletions, substitutions, and other structural modifications. These studies have expanded the knowledge of important viral processes such as cellular attachment, fusion, and entry and have concluded that retroviral Env glycoproteins can tolerate an enormous number of structural modifications and have elucidated the regions in which these modifications are viable. The results of these studies have lead to the development a number of transductional targeting strategies such as psuedotyping and targeting via ligand insertion of monoclonal antibody insertion.
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