As stated in its definition, a virus is a living organism that relies on the host machinery for its replication. Viral infection begins with the entry of the virus into the host target cell. In the absence of viral entry, productive infection cannot proceed, and there is an absence of viral replication. For this reason, virus entry machinery is an excellent target for antiviral therapeutics. In general, a virus life cycle includes these major steps: cell-surface attachment, entry, replication, assembly, and shedding, and for some viruses, latency. The early steps of the life-cycle refer to virus attachment, receptor binding and entry. These steps involve the initial interactions between a virus and the host cell thus is the major determinant of the tropism of the virus infection, the nature of the virus replication, the diseases resulted from the infection. Because of the pathological importance of these early steps for viral infectious diseases, they are important targets for antiviral discovery. In this review, Herpes Simplex Virus (HSV), Hepatitis C Virus (HCV) and human Enterovirus 71 (EV71) are used as representatives of enveloped DNA viruses, enveloped RNA virus and none-enveloped viruses. We summarize the current understandings of mechanisms of virus attachment and entry, and the strategies of antivirals screeings targeting these early steps of virus infection.
The General Theme of Virus Entry
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Enveloped viruses enter cells by fusion with plasma or endocytic membranes (Fig 1. ) (40). Membrane fusion is a complex process initiated by the specific interactions between host-cell-surface receptors and viral envelope glycoproteins(16; 27). Virus entry consists of three basic steps: recognition of cellular receptors by a viral glycoprotein, triggering of fusion and fusion execution. These steps are carried out and regulated by viral glycoproteins, in concert with their cognate receptors.
The surface of non-enveloped viruses is covered with capsid proteins(57). The capsid proteins of EV71, and picornaviruses in general, are encoded by the P1 region of the genome, and the capsid particles comprise 60 copies of four P1-encoded polypeptides, VP1 to VP4. The first three viral proteins (VP1-VP3) reside on the outer surface of the virus, while the shorter VP4 is located on the inner surface of the capsids(68; 81). The capsid proteins mediate the initiation of infection by binding to a receptor on the host membrane(59; 86; 87). Although non-enveloped viruses do not require membrane fusion for entry into cells, a membrane-binding motif is present in the capsid surface proteins(19; 28), which is responsible for activating host cell membrane to allow the entry of capsid.
The current understandings of the key viral and cellular components during the early steps of HSV, HCV and EV71 are summarized on Table 1.
HSV infection is one of the most common communicable diseases in humans, causing recurrent cold sores, keratoconjunctivitis, genital herpes and even life-threatening herpes encephalitis(72). Following entry into a host cell in the skin or mucosae, HSV undergoes primary replication. The virus then gains access to the distal axon terminals of sensory neurons and to sensory ganglia, where latent infection occurs(2). The initial replication phase, when the IE genes of HSV are expressed in the absence of viral protein synthesis, is critical. The IE proteins are required for subsequent viral protein expression and successful completion of the virus life cycle(48ï¼ŒBatchelor, 1990 #32). The current clinical management of HSV-related diseases uses chemotherapeutic agents mainly targeting the viral DNA polymerase, such as acyclovir(ACV) and (13; 25; 30; 55; 70), which are limited by the emergence of drug resistant viruses or their side effects(22; 52). It is estimated that 5% of the isolates from immunocompromised patients with HSV lesions have evidence of resistance(13; 25; 30; 55; 70). HSV infection is one of the most common sexually transmitted diseases, effective methods of prevention will be essential to their containment.
HSV is considered as the paradigm of herpesviruses with respect to virus entry into the cell. The HSV entry process is by far the most complicated, it requires number of viral glycoproteins function in concert to complete (21; 36; 69; 73). A HSV first attaches to cell membrane by interaction of gC and gB to heparan sulphate (HS) non-specifically. Since viruses recovered at this point remains it infectivity, it is unlikely that dramatic conformational changes resulted from such interactions (11; 42; 60; 67; 78). The second step of HSV entry requires the gD and the HSV entry receptor binding. Several HSV receptors have been identified, including nectin 1 and nectin 2, herpes virus entry mediator (HVEM, also named HveA for herpes simplex mediator A) and specific o-sulphates (3O-S) moieties in HS(31; 36; 73). The alternative usage and global expression of these receptors probably accounts for the entry of HSV into a wide range of different cell types. The third step of HSV entry is fusion of virion envelope with the plasma membrane of target cell. gB, gH, gL are required and constitute the conserved fusion machinery across the herpesvirus family(18; 21; 31; 36; 67; 69; 73). Molecular and biochemical analysis of gH suggest a class I fusion protein, however, its structure remain to be solved. The crystal structure of gB, on the other hand, exhibits a remarkable similarity to that of vesicula stomatitis G protein, and to viral fusion glycoprotein in general(3; 35; 84). The sequential interactions from among gD, gB and gH-gL have recently demonstrated, suggest that gD-receptor binding induces conformational changes of gB, which resulted in gB and gH-gL interaction prior to membrane fusion (4). How the two glycoproteins cooperate to execute fusion, and why two, and not one fusion executors are required in herpesvirus family is unclear. It is interesting to note that entry by fusion at plasma membrane, and entry by fusion in endocytic vesicle required all four glycoproteins (gD, gB, gH and gL), further details remain to be elucidated to different the individual characteristics of these two fusion processes.
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The World Health Organization estimated that at least 170 million people are currently infected with HCV, with 3-4 million new infections each year (43). HCV is one of the leading reasons for liver transplantation in the developed world, and numerous studies predict that the burden will continue to increase. At the present time there is no prophylactic vaccine against HCV, and the only approved treatment for infection is pegylated interferon and ribavirin with severe adverse effect. Clearly, new antiviral therapies are needed.
HCV enters cells by clathrin-mediated endocytosis(8; 37). Productive infection requires a low pH compartment and depends on the presence of cholesterol in the target cell membrane(17). Initial association of HCV with host cell surface involves nonspecific attachment to concentrate virions to subdomains of the cell surface(26). One identified attachment factor is heparin sulfate, a glycosaminoglycan(6; 34; 56; 88). HCV infection is inhibited in the presence of increasing amounts of soluble heparin, as well as by treatment of cells with heparinase I or III prior to infection(34). It is unclear whether heparin sulfate associates with HCV particles or the lipoproteins (e.g. low-density lipoproteins [LDL] and very low-density lipoproteins [VLDL]) commonly found in association with the virus(37). The LDL receptor is also implicated as an attachment factor, and adsorption of HCV particles can be inhibited by antibodies specific for either purified VLDL or its receptor(1; 89). C-type lectins, including DC-SIGN and LSIGN, are also implicated in HCV attachment(29; 50; 51; 80). However, the role for each of these attachment factors in HCV entry is unclear because they have not been shown to be required for productive infection. The role for DC/SIGN and L/SIGN in productive entry is uncertain, because they are not expressed on hepatocytes.
HCV E1 and E2 glycoproteins are responsible for interaction with cellular receptors/coreceptors. Increasing number of cellular factors have been implicated in HCV entry, including CD81, SR-BI, CLDN1, and highly sulfated glycosoaminoglycans (GAGs) (26; 37; 89). Since certain cell lines remain nonpermissive for HCV entry despite expression of all of these molecules, suggesting either additional unidentified entry factor(s) remain to be identified, or special mechanisms downstream from the receptor level are missing in these cell lines resistant to HCV entry(17; 66).
CD81 has four transmembrane domains, a small and a large extracellular loop (LEL). The LEL interacts with E2(90; 91). SR-BI also binds HCV E2(24). Contrary to CD81, SR-BI is highly expressed on hepatocytes to selectively uptake cholesterol and cholesterol esters from high-density lipoprotein (HDL) particles, thus enhance HCV infectivity(71). HCV infection is inhibited by CD81 and SR-BI antibodies(90; 91), RNAi targeting, protein fragments(83). Ligand of SR-BI, oxidized LDL, also inhibits HCV entry. CLDN1 is the other recently identified HCV entry receptor. It is a tight junction protein highly expressed in the liver and has four transmembrane domains and two extracellular loops. HCV entry requires residues within extracellular loop 1. HCVpp infection of 293T cells expressing CLDN1 remains CD81-dependent, further supporting the model that no one factor is sufficient for HCV entry (65).
Enterovirus 71 (EV71) is a small nonenveloped virus classified as Human enterovirus. It has an icosahedral capsid that enclose a single-stranded positive sense RNA genome. Its infection causes hand, foot, and mouth disease (HFMD) and herpangina(49; 81). EV71 infection is also associated with severe neurological diseases, such as brain stem encephalitis and poliomyelitis-like paralysis, mainly in infants and young children(61). Although the mechanistic understanding of EV71 entry is still very limited, its cell entry involves virus surface attachment, receptor binding, uptake by various endocytic pathways followed by release of the genome from the capsid (uncoating) and delivery of the genome across the endosome membrane into the cytoplasm(59; 86). There are evidences support the notion that cell surface sialic acid is important for initial viral attachment(87). Similar to enveloped viruses, the entry is initiated from the binding between the virus and the receptor. At physiological temperatures the formation of this initial complex induces conformational changes in the virus to form a tight-binding complex. Subsequent structural changes in the virus cause the viral capsid surface VP1 to move away from the fivefold axes, results in more extensive contacts with the receptor.
Two receptors of EV71 have recently been identified. Scavenger receptor class B member 2 (SCARB2)-a membrane protein previously implicated in the endocytosis of high-density lipoprotein and the internalization of pathogenic bacteria-is a functional receptor for EV71(86). Human P-selectin glycoprotein ligand-1 (PSGL-1)-a mucin-like protein involved in the tethering and rolling of leukocytes on vascular endothelium, is used by several EV71 isolates to infect lymphocyte cell lines(59). When expressed in mouse cells, PSGL-1 and SCARB2 each is sufficient for virus attachment and entry (86, Nishimura, 2009 #1). Since several EV71 isolates infect lymphocytes independently of PSGL-1, and SCARB2-specific antibodies can not completely block infection on some cell lines, additional receptor(s) remain to be identified. Furthermore, since the EV71-receptor interactions do not cause viral instability or uncoating, such interactions probably result in the aggregation of other receptors, or trigger the subsequent endocytosis(63).
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In spite of the major differences between enveloped and non-enveloped viruses, common themes have emerged in the membrane penetration processes of nonenveloped viruses, includes the presence of small, membrane active peptides in one or more of the capsid proteins(5; 15). These amphipathic or hydrophobic regions, while being analogous to the enveloped virus fusion peptides, cause membrane disruption rather than promoting membrane fusion. While host cell entry of non-enveloped viruses remains a diverse and largely unresolved area, current data indicates that the membrane active peptides of nonenveloped virus is comparable to that of fusion peptides in enveloped viruses(5; 15; 19; 28).
Strategies for Entry Inhibitor Screening
In the past years, assays specifically designed for the screening of inhibitors against the early steps of virus infection were developed. These assays are based on the understandings of virus entry mechanisms, and some of the assays are capable for robotic high-throughput-screening (HTS). With more detailed understanding of virus entry mechanisms, we anticipate more advanced assays being developed.
Recombinant Reporter Virus
For HSV, a recombinant HSV-1, HSV-1/Blue, which contains a HSV-1 ICP4 promoter directing a lacZ gene inserted into the HSV-1 TK gene was generated (Figure 2) (23). ICP4 promoter is a HSV immediate-early promoter, and could drive efficient LacZ expression within five hours after HSV-1/Blue infection (33), thus inhibition activities displayed using such assay are most likely due to the inhibition of the early steps of virus infection. In addition to HSV-1/Blue, there are a large number of different versions of recombinant HSVs were generated. Tanaka et al reported the construction of a recombinant HSV-1 (YK333) containing EGFP expression cassette driven by the Egr-1 promoter which is capable to detect antiviral activity within 48 hours post-infection (75).
For HCV, reporter viruses are only recently available when JFH-1-based infectious HCV clone was constructed. Recombinant viruses carrying reporter genes in their genomes, typically luciferase, or green fluorescent protein (GFP), are used for the measurement of viral RNA replication. These reporter viruses were either bicistronic or moncistronic, as illustrated in figure 3. A chimeric bicistronic JFH1 virus that carries the luciferase reporter gene in the viral cDNA sequence, was developed to characterize the early steps of HCV entry (41; 64). An analogous chimeric monocistronic reporter virus system was used to demonstrate time- and temperature-dependent activation of HCV for low-pH-triggered entry(79).
For EV71, reporter viruses were constructed using viral cDNA clones, however, there is no report of its application in entry inhibitor screenings (86).
Although often robust, especially for HSV, there are several limitations associated with screening systems with recombinant reporter viruses. For example, recombinant reporter HCV viruses are often attenuated and difficult to passage. In addition, the activities against primary viral isolates of the candidate inhibitors can not be answered using these recombinant viruses.
Virus Infection Reporter Cells
Another reporter system, which is complementary to the recombinant reporter virus system, is the reporter cell system. Such system either expresses a reporter protein (GFP or luciferase) under a virus infection activated promoter, or expresses a protein activated upon virus infection (and the expression of the viral protease).
For HSV, several reporter cell have been reported, such as Vero-ICP10-SEAP (82), or BHKICP6LacZ-5 (74). We have recently generated a HSV reporter cell line stably transfected with a HSV-2 ICP10 promoter directed luciferase (Luc) expression cassette (Figure 4). The ICP10 promoter is generally defined as an early promoter, but actually it can be regulated as an immediate-early promoter (85). This cell line could efficiently report within five hours after HSV infection.
For HCV, there are many versions of reporter cell systems developed; Most of these systems take advantage of the viral protease activity. The NS3/4A protease of different HCV genotypes recognizes the conserved NS4A/4B sequence DEMEEC-S/AXXX. Lee et al. constructed a fusion protein composed of EGFP) SEAP linked by an octapeptide spacer and the HCV NS4A/4B cleavage site (46; 47). This fusion protein could act as a substrate for the NS3/4A serine protease. The role of EGFP is to retain the entire fusion protein within the cell. Upon HCV infection, NS3/4A separates SEAP from EGFP-SEAP, which is further secreted into the extracellular culture medium. Recently, Iro et al. took this construct and generated a stable Huh7 cell line (39)(Fig. 5). The reporter cell line enables rapid and sensitive quantification of HCV infection, and quantifies virus entry efficiency.
For EV71, similar to that for HCV, the current reporter cell systems are built upon the viral protease activity. EV71 contains 2Apro and 3Cpro two proteases. Using the fluorescence resonance emission transfer (FRET) technology, an assay measuring the EV71 2Apro was designed(32). In this system, GFP2 and DsRed2 were connected in with the cleavage motif of 2Apro as a FRET pair. Upon protease cleavage in the context of virus infection, the separation of the tandem fluorophore substrate was monitored as a FRET disruption in real-time by fluorescent microscopy and in a quantitative fashion by fluorometry. Although remain to be done, such system could also be adapted for the 3Cpro. (14; 45)
Besides as a cell-based entry inhibitor screening assay, the combined applications of the reporter virus and the reporter cell system could also be used to determine the detailed working mechanism of the candidate compound. Using the HSV reporter systems as an example, as illustrated in figure 6, when the virus is allowed to mix with cells for attachment before a candidate agent is added (Fig. 6A), the assay would help to show if attachment is required. If an antiviral effect is observed only in this case, it indicates a post-attachment target for the candidate agent. Similarly, when the candidate agent is mixed with cells before testing virus is added (Fig. 6B), the assay would help to show if the agent functions before virus attachment to cells. If the agent functions, it means a cellular pre-attachment target; on the other hand, when the candidate agent is mixed with testing virus before adding to cells for infection (Fig. 6C), the assay would help to show whether the exposure of virus to the agent inactivates the virus before infection. If the virus is inactivated, obviously, the candidate agent has a viral pre-attachment target. Similarly, when the Vero cells in Figure 6 are replaced with Vero-ICP10-Luc, HSV-1/Blue could be replaced with any laboratory (or primary isolated) strains of HSV, while the inhibition efficacy could be quantified with the measurement of luciferase activity.
Pseudotyped viruses offer unique advantages for the screening of entry inhibitors of the virus from which the outer shell is derived. In addition to its safety and the easiness for experimental manipulation, it is the key surrogate approach for viruses difficult in cell culture, like HCV.
Retrovirus based pseudovirus system is the most used for HCV. HCVpp is considered the most biologically relevant reporter system for the study of HCV entry(12; 44; 62). It closely mimics the entry and serological properties of native HCV infection, such as the tropism for primary human hepatocytes and hepatocyte cell lines, pH dependence of the infection process, and neutralization by patient sera as well as monoclonal antibodies (mAbs) specific for E2(38) (7; 9; 54). The involvement of human CD81 in HCV entry was confirmed in this surrogate system(20; 58). HCVpp consists of HCV envelope glycoproteins assembled onto retroviral core particles carrying a reporter gene such as luciferase, green fluorescence protein (Figure 7). Use of HCVpp harboring a luciferase reporter permits easy detection of productive viral entry by the very sensitive luciferase assay.
In addition to the retrovirus based pseudotyping system, vesicular stomatitis virus (VSV) has also been used to generate HCV envelop pseudotyped virus. In this system, a VSV glycoprotein gene deleted virus (VSV--G) is used to infect a HCV E1E2 expressing cell to generate the pseudotyped virus(10; 76). This virus has been shown able to infect primary human hepatocytes.
Virus free cell-cell fusion
For some enveloped viruses, such as HSV and HIV, they express glycoproteins onto the plasma membrane of infected cells, which in turn can induce cell-cell fusion using similar mechanisms as the virus-cell fusion(53; 77). The cellular expression of these viral envelope glycoproteins has allowed for the measurement of membrane fusion events using cell-cell fusion or syncytia formation. Such system has been a powerful tool in helping to characterize the important attachment and fusion proteins and to identify entry inhibitors. This method has been enhanced by the addition of a reporter-gene system to the cell-cell fusion assay (figure 8). This improvement has provided a high-throughput and quantitative aspect to this assay, which can serve as a surrogate for virus entry and is therefore ideally suited in the screening of viral membrane fusion inhibitors.
Development of novel therapeutic molecules is an important challenge. In addition to the high mutation rate of virus genome, cellular toxicities and evasion of host defense systems represent the challenges caused by virus infection and replication, and highlight the importance of novel inhibitors in antiviral therapy. Regardless enveloped and non-enveloped viruses, virus attachment and entry inhibitors interfere with virus infection, prevent the initiation of virus replication. Experiences from well studied viruses like HIV demonstrate that the availability of assay systems allowing to specifically quantifying virus attachment and fusion are paramount to the development of such inhibitors.
Table 1. Current molecules involved in the early steps of HSV, HCV and EV71 infection
HS: Heparin Sulfate; SA: Sialic Acid
SCARB2: Scavenger receptor class B member 2
PSGL-1: P-selectin glycoprotein ligand-1
Figure 1. Life-cycle of a generalized virus infection. Early steps of virus infection include attachment, receptor binding and entry (via fusion with plasm membrane directly, or via endocytosis).
Figure 2. Recombinant reporter virus system. A recombinant reporter virus with a reporter protein (such as LacZ, or EGFP, or Luc, or SEAP) under a viral immediately promoter is used in this system. The level of virus entry is measured as the level of reporter protein expression.
Figure 3. HCV reporter viruses. A. A bicistronic reporter construct. B. A monocistronic reporter construct.
Figure 4. Virus entry assay based on reporter cells. A cell line with a reporter protein (such as LacZ, or EGFP, or Luc, or SEAP) under a viral immediately promoter is used in this system. The level of virus entry is measured as the level of reporter protein expression 5 hrs post infection.
Figure 5. Reporter cell based HCV infection assay. A EGFP-SEAP fusion protein linked with a HCV NS3/4A protease cleavage sequence is expressed in the reporter cell. Upon HCV infection and viral protein expression, EGFP and SEAP is separated via NS3/4A cleavage. SEAP is secreted to the culture media. The level of HCV infection is measured as the level of SEAP activity.
Figure 6. Determination of potential mechanisms of HSV entry inhibitors. Vero cell monolayer in 96-well plates are treated with HSV-1/Blue and a candidate entry inhibitor as shown in panels A, B, and C. If the maximum antiviral effect appears in A, the candidate agent has a post-attachment target, if in B, a cellular pre-attachment target, and if in C, a viral pre-attachment target.
Figure 7. Retrovirus pseudotyped with HCV E1E2.
Figure 8. Virus free fusion assay. Target cell: expresses virus receptor protein on its cellular membrane, and a reporter protein under T7 promoter. Effector cell: expresses viral entry glycoproteins and T7 polymerase. When target cell is cocultured with effector cell, membrane fusion is induced. T7 polymerase in effector cell will express the reporter protein expression. Level of membrane fusion is measured as the level of reporter protein expression.