Research Article: Retrovirus Entry by Endocytosis and Cathepsin Proteases

Date Published: December 6, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Yoshinao Kubo, Hideki Hayashi, Toshifumi Matsuyama, Hironori Sato, Naoki Yamamoto.


Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines.

Partial Text

Retroviruses include many pathogenic agents in humans and animals. Human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) induce acquired immunodeficiency syndrome (AIDS) and adult T-cell leukemia (ATL), respectively. Murine leukemia viruses (MLVs) are also well-studied among retroviruses because the MLVs are used comparatively as animal models of several human diseases (leukemia, immunodeficiency, and neuropathogenic diseases) and as gene transfer tools. In addition, there are animal retroviruses that are important problems in the livestock industry, such as Visna, equine infectious anemia virus, bovine leukemia virus, and Jaagsiekte sheep retrovirus.

Mechanism of membrane fusion by the retroviral TM proteins is described elsewhere in details [3–7] and is similar to those used by envelope proteins of other enveloped viruses [8, 9]. Briefly, the retroviral entry mechanism is proposed as follows. The TM protein is thought to have hairpin-like structure (Figure 1). The binding of SU with its cognate cell surface receptor induces conformational changes of the TM subunit. The N-terminal hydrophobic domain of the TM subunit called fusion peptide is exposed by the conformational change and inserted into host cell membrane. The TM protein then coverts to a trimer-of-hairpins conformation, and viral envelope and host cell membranes approach and mix. Finally, the fusion pore is formed and expanded to derive the viral core into host cell cytoplasm. This conformational change pathway of the TM protein induces the membrane fusion for the retroviral entry into host cells.

In this section, we will mainly focus on the infection receptors for MLV and HIV, with which entry mechanisms are most extensively studied among retroviruses. Other reviews should be referred to concerning the infection receptors of animal retroviruses in general [10, 11]. MLVs are divided into four groups according to their host ranges and infection interference, and the four groups recognize different cell surface receptors. Ecotropic MLVs infect mouse and rat and bind to cationic amino acid transporter 1 (CAT1) as the infection receptor [12]. Amphotropic MLVs infect many types of mammals, and inorganic phosphate symporter 2 (Pit2) is the amphotropic infection receptor [13, 14]. Polytropic MLVs has a similar host range to the amphotropic MLVs. The amphotropic MLVs cannot infect amphotropic virus-infected cells, because Pit2 are already occupied by the amphotropic Env proteins, called infection interference. Whereas the polytropic MLVs can infect amphotropic virus-infected cells, indicating that the polytropic virus receptor is different from the amphotropic receptor. Polytropic MLVs recognize XPR1 for the infection [15–17], whose physiological function is unknown yet. Xenotropic MLVs recognize the XPR1 as polytropic MLVs, but do not infect mouse cells. These MLV infection receptors are all multimembrane spanning proteins.

When retrovirus-producing and -susceptible cells are mixed, viral Env proteins on the cells can effectively interact with infection receptors on the neighboring susceptible cells via direct cell-to-cell contact. The interactions can have both positive and negative effects on the retrovirus replication. First, they can lead to cell-to-cell infection that allows very rapid and synchronized replication of virus compared to the cell-free infection [25, 26]. This can be advantageous for the virus replication in the presence of antiviral agents [27]. Second, the interactions can induce a negative effect, that is, the rapid apoptotic cell death, via syncytium formation [28–30]. This can be disadvantageous for the virus in that the sustained production of progeny virions becomes impossible. If the apoptotic cell death proceeded more efficiently than the virus replication, it eventually would result in poor progeny virus production. Therefore, it is conceivable that the retroviruses have some mechanisms to attenuate fusion capability of the envelope TM proteins in virus-producing cells and to primarily activate it in retroviral particles upon virion budding. Consistently, such mechanisms have been suggested for the Env TM proteins of MLV and HIV.

Ammonium chloride, a weak base, neutralizes acid conditions in intracellular vesicles (Table 1). Concanamycin A and bafilomycin A-1 are specific inhibitors of the ATP-dependent proton pump/vacuolar ATPase (V-ATPase) that serves to acidify endocytic vesicles [46, 47]. To analyze the pH dependence of retrovirus entry, these compounds are frequently used. Additionally these inhibitors may affect trafficking of the intracellular vesicles, because siRNA-mediated knockdowns of subunits of V-ATPase complex affect trafficking of intracellular vesicles [48]. Previously it had been reported that ammonium chloride inhibits ecotropic MLV infection but does not amphotropic and xenotropic MLV infections, showing that ecotropic MLV infection occurs through acidic vesicles, but amphotropic and xenotropic MLV infections do not [49, 50] (Table 2). The more specific inhibitors of endosome acidification (concanamycin A and bafilomycin A-1) suppress all of ecotropic, amphotropic, polytropic, and xenotropic MLV infections [51, 52]. At present, it is generally accepted that ecotropic MLV infection requires acidification, because all the studies consistently reported the suppression of ecotropic virus replication with the inhibitors of endosome acidification. In contrast, it has been shown that xenotropic MLV infections are not suppressed by bafilomycin A-1 [53] (Table 2). Due to the controversial results, the entry pathway of xenotropic MLV is not clear yet. Because different cell lines were used in those reports, the low pH requirement of the xenotropic MLV infection may be dependent on the used cell lines (see below).

The requirement of low pH for the retrovirus infections reveals that retrovirus particles are internalized into acidic intracellular compartments during virus replication. There are several different pathways for the internalization of molecules; (i) phagocytosis, (ii) macropinocytosis, (iii) clathrin- and dynamin-dependent endocytosis, (iv) caveolin- and dynamin-dependent endocytosis, (v) lipid raft- and dynamin-dependent endocytosis, (vi) clathrin-, caveolin-, and dynamin-independent endocytosis that requires lipid raft, and (vii) dynamin-, clathrin-, caveolin-, and lipid raft-independent endocytosis [48, 71]. Here we will briefly summarize the accepted mechanisms and roles of internalization, relevant to the present review [48, 72, 73].

A dominant negative mutant of caveolin [74], siRNA-mediated knockdown of dynamin, and a dynamin inhibitor (dynasore) (Table 1) [52] suppress the amphotropic MLV infection, suggesting that amphotropic MLV particles are internalized by the dynamin- and caveolin-dependent endocytosis for productive infection (the fourth pathway). Ecotropic MLV particles are internalized into intracellular vesicles, but the vesicles are not colocalized with clathrin [75]. Furthermore, the dynamin-dominant negative mutant does not inhibit ecotropic MLV infection in human HeLa cells expressing the ecotropic MLV receptor, suggesting that ecotropic MLV particles are internalized by clathrin- and dynamin-independent endocytosis [75]. In contrast, another report indicates that siRNA-mediated knockdown of dynamin and dynasore suppresses ecotropic MLV infection in mouse NIH3T3, rat XC, and human TE671 cells expressing the ecotropic receptor [52] (Table 3). As mentioned above, the internalization pathway of ecotropic MLV might be dependent on the cell lines used. ALV [76] and EIAV [77] infections occur through clathrin-dependent endocytosis. JSRV infection required dynamin-dependent endocytosis [69]. Taken together, these reports strongly support a notion that infections by many animal retroviruses occur through endosomes and require endosome acidification.

Many retrovirus infections require endosome acidification. Influenza virus infection also requires endosome acidification, and treatment of influenza virus particles with low pH buffer activates its membrane fusion, indicating that low pH treatment directly induces conformational change of the influenza virus hemagglutinin to the fusion-active form. In contrast, low pH treatment of MLV particles does not activate the membrane fusion. Why does ecotropic MLV entry require endosome acidification?

Although the acidification inhibitors attenuate the ecotropic MLV infection in almost all susceptible cells [49, 52], the inhibitors have no effect on the ecotropic MLV infection specifically in rat XC cells, suggesting that the ecotropic MLV infection in XC cells is independent of low pH [49] (Table 2). Furthermore, the R peptide-containing ecotropic Env protein can induce pH-independent syncytium formation in XC cells, but cannot in other susceptible cells [82, 83]. By these results, it had been widely thought that ecotropic MLV entry into XC cells occurs at cell surface membranes and does not require the internalization of virions into intracellular vesicles and acidification. This XC cell-specific pH-independent ecotropic MLV infection was one of the well-known mysteries in the MLV field [84, 85]. We found that a cathepsin inhibitor, CA-074Me, efficiently suppresses the ecotropic MLV infection in XC cells, like in other susceptible cells, suggesting that the ecotropic MLV infection in XC cells requires endosomal cathepsin proteases [52]. This result is inconsistent with the previous theory that the ecotropic MLV infection in XC cells does not occur through endosomes. Because the ecotropic MLV infection requires cathepsin proteases activated by endosome acidification, the acidification inhibitors would be proposed to suppress the MLV infection by attenuating cathepsin activation. However, the acidification inhibitors do not reduce cathepsin activity in XC cells, but do so in other cell lines, suggesting that cathepsin proteases are activated without endosome acidification in XC cells [52]. XC cells do not express so much cathepsin that activation is sufficient at suboptimal pH, because cathepsin activity of XC cells is comparable to that of NIH3T3 cells. These results prompted us to speculate that the ecotropic MLV infection in XC cells occurs through endosomes. The result that dynasore and siRNA-mediated knockdown of dynamin expression suppress the ecotropic MLV infection in XC cells strongly supports this hypothesis.

The R peptide-cleaved MLV Env protein induces the fusion between the viral envelope and host cell membranes for viral entry and syncytium formation in susceptible cells [33, 34]. Cells expressing the R peptide-truncated Env protein behave as large MLV particles and fuse with neighboring susceptible cells. Therefore, the syncytium formation by the retroviral Env proteins is thought to represent the membrane fusion in retroviral entry. Because the syncytium formation by the retroviral Env protein may contribute to the development of degenerative disorders like AIDS [28, 29], and because an endogenous retroviral Env protein (syncytin) induces syncytiotrophoblast formation [86], the elucidation of mechanism of retroviral Env-induced syncytium formation is essential to understand retroviral pathogenesis and placenta development. The MLV entry into host cells is dependent on low pH, but the syncytium formation by the R peptide-truncated Env protein is independent [33]. Furthermore, the viral envelopes fuse with host cell membrane in endosomes [52, 75], but the syncytium formation appears to result from the fusion of cell surface membranes of the Env-expressing and host cells. In addition, the Env glycoprotein of a CD4-independent HIV efficiently induces pH-independent syncytium formation [87], but infection by CD4-independent HIV occurs through acidic endosomes [21] (see below). Multiple interactions between the viral Env and infection receptor proteins in much larger areas of cell-cell contact than virus-cell contact may abrogate the requirement of endocytosis for the membrane fusion. The finding that a cell adhesion molecule, LFA-1, facilitates HIV-mediated syncytium formation but not HIV infection supports this idea [88]. If the syncytium formation by the Env protein is independent of endocytosis, cathepsin proteases would be unnecessary for the syncytium formation. However, cathepsin inhibitors suppress syncytium formation by the ecotropic MLV Env protein [79]. Secreted cathepsin proteases may be involved in the pH-independent syncytium formation by the Env protein. Further study is needed to understand the mechanism of pH-independent syncytium formation by the retroviral Env proteins.

There are many controversial reports of the role of endocytosis in CD4-dependent HIV infection [94] (Tables 2 and 3). Early reports indicate that the acidification inhibitors enhance [89–91] or do not affect CD4-dependent HIV infection [92, 93], suggesting that the HIV does not enter into host cells via acidic vesicles. However, recent reports show that dynasore and chlorpromazine attenuate CD4-dependent HIV infection [95–97]. In addition, dominant negative mutants of dynamin and Eps15 inhibit CD4-dependent HIV infection [98]. Furthermore, analysis of localization of labeled HIV particles revealed that the HIV particles are internalized into intracellular vesicles [95, 99–102]. It has been reported that envelopes of HIV particles fuse with host cell membranes in intracellular vesicles by the following observation [95]. Envelopes of HIV particles were labeled with a hydrophobic fluorescent compound. When fusion of the labeled HIV envelope with host cell membrane occurs, the fluorescent compound is diluted and the fluorescent signals disappear. The vanishing of the fluorescent signals was observed in the intracellular vesicles but not at cell surfaces. These results suggest that HIV entry into the host cell cytoplasm may occur via endosomes.

Because acidification inhibitors enhance CD4-dependent HIV infection [89–91], HIV entry is independent of low pH, and the viral particles internalized into acidic late endosomes are degraded [105]. In other words, a proportion of HIV particles are internalized into acidic late endosomes although the internalization into late endosomes is not associated with the HIV productive infection. Consistently, the HIV particles appear to be internalized into acidic compartments shortly after inoculation into host cells [100].

Retroviral vectors are valuable tools in molecular biology research and human gene therapy. Several fundamental properties of retroviral vectors remain to be improved for effective gene transfer to specific target cells [114]. The effectiveness will be greatly enhanced, if their infection tropism is artificially modified to target specific cells [115]. There have been various attempts to establish redirecting infection tropism by genetically incorporating heterogenous ligands into the retroviral Env proteins [116–121]. However, retroviral vectors containing such modified Env proteins suffer from very low transduction efficiency or are not infectious. The redirected transductions of retroviral vectors with chimeric Env proteins are enhanced by the endosome acidification inhibitors, suggesting that the targeted vector particles internalized into acidic endosomes are degraded by endosome proteases [120, 122].

Retrovirus vectors can be pseudotyped with glycoproteins of various enveloped viruses. The pseudotyped retrovirus vectors enter into host cells by the entry mechanisms of the heterologous viral glycoproteins. Because the retrovirus vectors do not produce replication-competent viruses and the protocol is relatively simple, pseudotyped retrovirus vectors are widely used to identify entry pathways of various enveloped viruses [126–128].

Infections by many animal retroviruses occur through endosomes and require endosome acidification. The activation of cathepsin proteases by endosome acidification is required for ecotropic MLV infection. Whereas acidification directly induces conformational changes of several retroviral Env proteins to the fusion active forms. There are several internalization pathways of retrovirus particles, and the viral internalization pathways appear to be different in different cell lines. CD4-independent HIV infection may occur through endosomes and require endosome acidification, like other animal retroviruses. CD4-dependent HIV infection is thought to occur through endosomes but does not require endosome acidification. The CD4-dependent and -independent HIV particles are both degraded by endosome proteases, when the viral particles are internalized into acidic late endosomes. Retrovirus vectors pseudotyped with other viral envelope proteins are widely used to understand the entry mechanisms of the envelope proteins. However, entry pathway(s) of the pseudotyped retroviral vector could be different from that of the original virus.