The heretofore unstudied protein I2 is encoded by one of the 90 genes that is conserved in all chordopoxviruses. I2L is predicted to encode a protein of 73 aa, and we have verified that the protein is indeed expressed at late times of infection. Using a recombinant virus encoding HA-tagged I2 from the endogenous locus, we also showed that the I2 protein is packaged in mature virions; fractionation studies indicate that I2 is found in the membrane of purified virions. Given our results, it is somewhat surprising that I2 was not identified as a virion component in recent studies profiling the composition of virions by mass spectroscopy (6
). We attributed this discrepancy to the fact that I2 is a very small protein and may also be present at low copy number. The I2 protein can be extracted from the virion membrane with NP-40 alone, as can the A13 protein, which is also a very small protein that contains a single membrane-spanning domain (32
). In contrast, the A14 and A17 proteins, which form inter- and intramolecular disulfide bonds and each span the membrane twice, can only be extracted with NP-40 and DTT (32
). The integral association of I2 with membranes in vivo and the incorporation of I2 into the virion membrane are likely to be mediated by the extremely hydrophobic domain present at the C terminus of the protein. Because this domain is required for stable expression of the protein, we could not directly test the hypothesis that it serves as a membrane targeting domain for I2.
To address the function of I2 in the viral life cycle, we generated an inducible recombinant in which the expression of I2 from its own promoter was dependent upon inclusion of TET in the culture medium. When I2 was repressed, gene expression, DNA replication, and genome maturation proceeded normally. Moreover, core proteins appeared to undergo normal proteolytic processing, and electron microscopic analysis confirmed that the full spectrum of morphogenetic intermediates was present. Mature virions were readily detected, as were wrapped and enveloped viruses. However, the yield of infectious virus at 24 hpi was reduced by 100- to 1,000-fold in the absence of I2. Virion production was not compromised, but the particle/PFU ratio of the I2-deficient virions was ~400 fold greater than that of those containing I2. Nevertheless, these I2-deficient virions had a wt appearance and contained a normal complement of protein and genomic DNA.
The infectivity defect seen with the I2-deficient virions manifested itself at an early stage. These virions did not induce any CPE, nor were they able to direct the synthesis of early proteins. Furthermore, they were also unable to mediate “fusion from without”: cells inoculated with I2−
virions did not induce syncytia after being pulsed with pH 5.5. Acid-induced syncytium formation is now known to mimic viral entry: the brief treatment with low pH mimics the environment presented by the endosomal pathway of entry/fusion that can be utilized efficiently by poxvirus particles (51
). This suggestion that I2-deficient virions were defective in virion entry was reinforced by immunofluorescence analysis, which verified that I2-deficient virions retain the ability to bind to target cells but lack the ability to enter cells, as defined by deposition of subviral cores within the cytoplasm.
Binding to target cells is mediated, at least in part, by the association of several virion proteins (A27, H3, and D8) with GAGs on the cell surface as well as the interaction of the A26 protein with laminin (5
). There is a presumption that a higher-affinity and/or more specific interaction must take place between as-yet-unknown virion proteins and cellular receptor(s). Neutralizing antibodies have been generated to the H3, A27, and L1 proteins. H3 and A27 are not essential for the production of infectious virus, making them unlikely candidates for receptor-binding proteins. The possibility that L1 may play this role has been difficult to test, because repression of L1 has been shown to block the assembly of mature virions (40
). Thus, we cannot be sure how virions that are positive for GAG and laminin interactions, but negative for receptor interactions, would score in the virion binding assay. We are therefore following convention in saying that I2-deficient virions can bind to cells, but we cannot rule out that the possibility that I2 may indeed contribute, either directly or indirectly, to the establishment of a “binding state” that may be a prerequisite for entry.
Production of viral particles that bind to, but cannot enter, cells has recently been associated with mutations in numerous vaccinia virus genes. The A16, A21, A28, G3, G9, H2, J5, L5, and F9 proteins have been shown to be dispensable for virion morphogenesis and cell attachment but essential for core deposition into the host cell (3
). A conserved feature shared by all but one of these proteins (G3) is the presence of disulfide bonds formed by the virally encoded redox machinery. Eight of these proteins (all but F9) have been shown to associate in a multiprotein complex now known as the EFC (47
). The role of individual proteins within the complex is just beginning to be deciphered, but it is known that the EFC does not form in the absence of the virion membrane, nor does it form if either A21 or A28 are absent. H2 and A28 are known to interact directly (35
). The F9 protein associates with the EFC at substoichiometric levels but is not required for EFC formation (3
). F9 shows significant sequence similarity to the L1 protein, which was previously found to play a role in virion morphogenesis (40
). However, very recent data show instead that L1 is dispensable for virion morphogenesis but, like F9, associates with the EFC and is essential for virion entry (2
The EFC has also been shown to associate with a heterodimeric complex containing the A56 (HA) and K2 proteins, which are known as fusion regulatory proteins. This interaction is mediated by A16 and G9 (62
). In the absence of either A56 or K2, infected cells form syncytia spontaneously at neutral pH. These data led to the hypothesis that the A56/K2 proteins prevent syncytium formation by interacting with A16/G9 and blocking EFC-mediated fusion. It seems reasonable to propose that, during the entry of wt viruses, the EFC-A56/K2 interaction is disrupted in the acidic environment of the endosome, activating fusion of the virion and vesicular membranes and resulting in core release.
The data presented here indicate that the I2 protein is the 10th protein that is required for virion entry. Although I2 was not identified as part of the EFC, its presence within the EFC is still possible. It may have been overlooked because of its small size (molecular weight of 8,000), or it may, like the F9 and L1 proteins, be present in substoichiometric levels (3
). A fivefold reduction in the levels of some of the EFC proteins was observed within I2-deficient virions, suggesting that I2 might play a role in the stabilization of the EFC or in recruitment of EFC proteins to nascent virion membranes. This 5-fold reduction in EFC proteins seems unlikely to account for the 400-fold decrease in the specific infectivity of the I2-deficient virions. However, it may be that the fivefold reduction does indeed bring the level of the EFC below a threshold needed for efficient entry; it is also possible that the reduction in EFC proteins is greater than our immunoblot analyses indicate. Alternatively, I2 might contribute to virion entry in an EFC-independent manner.
The small size of the I2 protein and the absence of disulfide bonds make I2 quite distinct from the entry proteins that comprise the EFC. One of the striking features that emerges from comparative analysis of the I2 orthologs present in diverse poxviruses is the C-terminal hydrophobic domain. We propose that this region is a membrane targeting domain (MTD) that, like the MTDs of the poxviral H3 protein (11
) and other tail-anchored proteins, mediates posttranslational insertion of I2 into nascent virions, although cotranslational insertion is also possible. The N-terminal portion of the protein contains several highly conserved regions. Results from a limited structure/function analysis revealed that three clusters of charged residues within this portion are not essential for I2's biological function. However, the highly conserved N-terminal 12 aa, which are also hydrophobic in character, are required. Similarly, perturbation of a small hydrophobic/aromatic cluster (FI23
) within this N-terminal domain renders I2 biologically inactive. The hydrophobic and aromatic residues within the N-terminal domain of I2 may mediate protein-protein interactions within the virion membrane or between the virion and the target cell. Alternatively, the N terminus of I2 may participate directly in membrane fusion.
The process through which poxviruses enter cells has recently become an area of intense study. First, it seems striking that at least 11 viral proteins are required for entry. Second, new insights into the mechanism of entry itself are emerging rapidly. Historically, fusion of the virion membrane with the plasma membrane was thought to occur (4
). More recently, it has emerged that the majority of virions are internalized into an endosomal compartment, with subsequent membrane fusion and cytoplasmic core deposition occurring only upon compartment acidification (51
). The initial internalization of virions has recently been shown to occur via macropinocytosis, a process which is upregulated when the binding of virions to cells induces PAK1-mediated signaling events that result in massive cell blebbing (31
). Macropinocytic uptake requires the presence of phosphatidylserine in the virion membrane, suggesting that virion uptake mimics the engulfment of apoptotic fragments. The presence of cholesterol in the plasma membrane of the target cell has also been shown to be important for virion entry (8
Deposition of the subviral core within the cytoplasm requires fusion of the virion membrane with the membrane of an endocytic compartment (or, less frequently, with the plasma membrane). Because none of the EFC proteins has been shown to have fusogenic activity, we have no insight into how this process occurs. The viral A17 protein has been shown to mediate cell-cell fusion when expressed at high levels on its own in insect cells and to do so when coexpressed with the A27 protein in mammalian cells (26
). Because A17 is essential for the early stages of virion morphogenesis (28
), it has not been possible to test its role in virion entry. Thus, a role for A17 as a mediator of fusion in the context of infection remains to be formally tested.
The intricacy of the poxvirus life cycle has proven to be illuminating for virologists, immunologists, and cell biologists alike. Recent studies have shown that process of poxvirus entry is surprisingly complex, and further study of how I2 collaborates with many other viral proteins to facilitate the initial interactions of poxvirus virions with their target cells should prove to be of great interest.