The replication cycle of picornaviruses occurs entirely in the cytoplasm. Infection comprises many processes that are poorly understood, including the induction of increased phospholipid synthesis, a block in protein secretion from the ER and Golgi complex, the formation of cytoplasmic vesicles which harbor the viral replication complex, and alterations in intracellular calcium content in the cell (5
). While the viral proteins responsible for some of these changes have been identified, their mechanisms of action are not known. Many consequences of viral infection are likely to be effected by interactions between viral and host cell proteins. Despite this assumption, very few such interactions have been identified.
To identify viral proteins that might interact with cell proteins, we selected a variant of HRV39 that replicates in mouse cells. Although replication of this serotype cannot be detected in mouse cells, stocks of this virus may contain low levels of viral variants that are able to grow in mouse cells. These variants are presumably selected upon passage in ICAM-L cells. The genome of the mouse-adapted virus RV39/L encodes amino acid changes in both the 2B and the 3A proteins. Previously, human rhinovirus 2 (HRV2), a minor-group serotype of rhinovirus related to HRV39, was adapted to mouse cells (81
). In infected cells, the adapted virus RV2/L produced P2 proteins that migrate with altered mobility on protein gels compared with P2 proteins of HRV2. Nucleotide sequence analysis of RNA encoding 2ABC3AB revealed 10 coding changes in the genome of RV2/L, three of which are identical to changes seen in RV39/L (V2I and K36E of 2B and I35M of 3A) (Fig. ) (F. Yin, personal communication). RV2/L also exhibited altered sensitivity to inhibitors that specifically block viral RNA replication, leading the authors to suggest that the process of viral RNA replication was altered in an unknown manner. The influence of the individual amino acid changes of RV2/L in the adaptation to mouse cells has not been determined. The same group subsequently isolated mouse-adapted variants of HRV39 by transfection of viral RNA in L cells (46
). Of three independent virus stocks used, only one produced infectious virus after RNA transfection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that a P2 protein of the virus that replicated in mouse cells migrated differently from that of the other two virus stocks. Nucleotide sequence from the adapted virus was not obtained. The authors suggested that a P2 protein was an essential viral component that could function in mouse L cells only in an altered form and that genetic changes leading to this altered form arose spontaneously during viral growth in permissive human cells (46
). A P2 protein was also implicated in the adaptation of HRV16 to growth in mouse cells (33
). Amino acid changes needed for replication of HRV16 in mouse cells are located in protein 2BC and appear to cause conformational changes in the viral protein that may influence interaction with a host cell protein.
To identify the specific protein(s) involved in our adaptation of HRV39 to mouse cells, viruses with the amino acid changes of RV39/L in either the 2B or 3A protein were produced (Fig. ). The results of one-step growth curve analysis in ICAM-L cells indicated that RV39/IGE replicated with kinetics similar to those of RV39/L (Fig. ). While the titers of both RV39/IGE and RV39/L reached similar levels by 12 h postinfection, the titer of RV39/IGE did not exceed the starting titer, while RV39/L titers increased 10-fold. These differences were reflected in the results of RNA analysis: at 12 h postinfection, the amount of RV39/IGE RNA did not exceed starting levels, while the amount of RV39/L viral RNA increased (Fig. ). However, despite the large difference in viral RNA levels at 12 h postinfection, titers of RV39/L and RV39/IGE were nearly identical. The reason for this observation is not known, but one possible explanation is that RNA packaging or assembly of RV39/L virions is affected by the amino acid changes in 2B and/or 3A. Overall, the growth curve and RNA analyses indicate that, while the amino acid changes in 2B contribute significantly to the adaptation to mouse cells, the additional changes in 3A are required for complete adaptation. In particular, changes in the K36 residue of 2B seem to be important in mouse cell adaptation of HRV39. The similar growth in human cells of viruses with amino acid changes in either the 2B or 3A protein confirmed that the adaptation was specific to mouse cells (Fig. ). Analysis of viral RNA production in mouse cells demonstrated that amino acid changes in both 2B and 3A were required to achieve comparable levels of viral positive-strand genome production to that seen in cells infected with RV39/L. RNA levels could not be determined at later times postinfection due to the presence of substantial cytopathic effect.
There are two hydrophobic regions in 2B that are both important in the functions of the protein (75
) (Fig. ). HR1, the more amino-terminal hydrophobic region, is a cationic amphipathic α-helix thought to span the membrane. The second hydrophobic region, HR2, is a transmembrane domain. Both HR2 and the hydrophilic region between HR1 and HR2 have been shown to be important in multimerization of coxsackievirus B3 2B (21
). This viral protein localizes to the Golgi complex in COS-1 cells: deletion of HR1 changes the localization to the ER (22
), although HR2 is also important in proper localization. Other studies have demonstrated that 2B localizes to the ER and the Golgi complex (58
). The properties of 2B suggest that it functions as a viroporin, oligomerizing and inserting into membranes to create pores (31
). The formation of these pores may explain the increase in intracellular calcium observed in the cytoplasm of infected cells by allowing the passage of calcium from either the ER lumen or the extracellular milieu (37
). Another known effect of 2B (and 2BC) synthesis is to block protein export from the Golgi complex (8
). At least the N-terminal 30 amino acids of 2BC are likely to be involved in this function (7
). Alterations in the 2B protein that impair virus growth but do not affect the ability of the protein to permeabilize membranes or block protein secretion have been described, suggesting additional functions for 2B (77
Three amino acid changes were identified in the 2B protein of our mouse-adapted RV39/L, including one amino acid change—K36E—in HR1. This residue is a glutamate in several of the picornaviruses shown in Fig. . Of the viruses with a glutamate residue at this position, all are able to grow in mouse cells (2
), suggesting that this residue may be critical in determining host range. This amino acid is also altered from a K to an E in mouse-adapted HRV2/L (F. Yin, personal communication). The other two amino acid changes in protein 2B of RV39/L, V2I and E11G, do not appear to exhibit any particular pattern in the other viruses that would suggest an obvious role in host range (Fig. ). It seems unlikely that any amino acid change involved in host range expansion would affect a critical function such as multimerization, pore formation, or inhibition of protein secretion. However, these activities may require a host protein, and the K36 residue could be critical in the interaction between 2B and a host protein.
Considerable evidence implicates the 3A protein and its precursor, 3AB, in RNA replication (10
). A strongly hydrophobic region in the C terminus of 3A is involved in membrane association and is likely to mediate inclusion of 3A in the replication complex (45
) (Fig. ). Recently, the solution structure of the soluble N-terminal domain of poliovirus 3A was determined, showing that 3A forms homodimers with unstructured N- and C-terminal regions in each monomer (60
). The G41 residue of poliovirus 3A, immediately following the last residue in the second of two amphipathic α-helices, corresponds to the K33 residue of rhinovirus 39 3A (Fig. ). The W42 residue of poliovirus 3A, which acts in concert with a number of other N-terminal residues to “bury” the dimer interface, is directly adjacent to the rhinovirus I35 residue. While these regions are not directly involved in dimerization of poliovirus 3A, they do contribute to 3A structure. It was suggested that long-range contacts between G41 or its neighbor, W42, and N-terminal residues mediate the structure of poliovirus 3A. Given these data, it seems likely that the amino acid changes observed in RV39/L 3A cause conformational changes in the protein that may alter its interactions with host cell proteins or other viral proteins. Evidence for possible interaction between 2B and 3A comes from studies of chimeric viruses with the hydrophobic domain of poliovirus 3A exchanged for that of rhinovirus 3A. These chimeric viruses are viable but do not replicate as well as wild-type virus. Viruses with improved replication were isolated that contain compensatory changes in the 2B protein, suggesting that 2B and 3A may interact during infection (71
). Recent data also suggest interactions between 2B, 2C, and 3A (65
We observed selection for an amino acid change at residue K36 of 2B protein during growth of HRV39 in mouse cells (Table ). However, changes in 2B do not fully account for the adaptation of RV39/L to mouse cells: RV39/IGE does not grow as well as RV39/L in mouse cells, nor does it produce the same amount of RNA as RV39/L by 12 h postinfection (Fig. ). Although RV39/RM did not replicate as well as RV39/IGE, in the presence of the 2B changes, alterations in 3A clearly enhance viral replication in mouse cells. How do these changes facilitate adaptation to mouse cells? It is possible that the interaction of 2B (or a 2B precursor) with a host cell protein mediates growth in mouse cells. If contacts also occur between 2B and 3A, amino acid changes in 3A could facilitate improved interactions with 2B protein of mouse-adapted RV39/L. We were unable to demonstrate interaction between HRV39 2B and 3A using the yeast two-hybrid system (data not shown), but this result should not be considered definitive evidence of a lack of interaction during infection. Alternatively, 2B and 3A might interact with the same host cell protein, or with two different proteins. In either case, the interaction of 2B with its putative cellular partner would have a greater influence on growth in mouse cells than the interaction of 3A and its cellular partner.
Most of what is known about virus-induced alterations of cell membranes comes from studies of poliovirus, the prototype picornavirus. The most drastic structural change observed in poliovirus-infected cells is the loss of intact ER and Golgi complex and the accumulation of membranous vesicles in the cytoplasm (18
). Most of the nonstructural proteins and some nonstructural protein precursors have been found on the surface of these vesicles (12
). The synthesis of 2BC and 3A in uninfected cells leads to the formation of vesicles identical in buoyant density and appearance to those present during poliovirus infection (61
). The vesicles include markers from different organelles, including the ER and the Golgi complex (23
). The requirement for de novo phospholipid synthesis for poliovirus replication suggests that vesicle membranes may be newly synthesized rather than exclusively derived from preexisting membranes (32
There is controversy over the origin of the vesicles induced by poliovirus infection. Many of the vesicles found in poliovirus-infected cells are double membraned, originate from multiple organelles, and have cytosolic content, suggesting a mechanism of formation similar to cellular autophagy (59
). However, recent evidence demonstrated that poliovirus-induced vesicles are morphologically similar to COPII vesicles, which are involved in anterograde transport (57
). The COPII proteins Sec13/31 were shown to colocalize with the viral 2B protein on the surface of the vesicles. The authors found that viral vesicles bud from the ER but do not fuse with the ER/Golgi intermediate complex and suggested that the cellular COPII machinery participates in the induction of vesicles suitable for poliovirus replication. However, the inhibition of poliovirus and rhinovirus RNA replication by brefeldin A directly contradicts this hypothesis (20
). Brefeldin A inhibits formation of COPI but not COPII vesicles. In addition, overproduction of dominant-negative Sar1, a COPII component, was found to block anterograde transport but not poliovirus replication (63
). These observations argue against a COPII origin for poliovirus vesicles.
The vesicles formed by HRV39 in ICAM-L and HeLa R19 cells appear different from each other when examined by electron microscopy. The vesicles in HeLa R19 cells are densely packed, similar to vesicles observed during infection with other picornaviruses (12
). The vesicle shape is elongated and irregular, and the vesicles appear to contain electron-dense material. Vesicles induced in ICAM-L cells are mostly round, larger, and more dispersed. Most of the rhinovirus-induced vesicles appear to have single membranes, in contrast to poliovirus-induced vesicles. However, the formation of predominantly single-membraned vesicles has been reported for other picornaviruses (49
), and vesicles of this morphology have also been observed during poliovirus infection (61
Despite the absence of detectable HRV39 RNA replication in mouse cells (Fig. ), vesicles are formed during HRV39 infection (Fig. ). This observation suggests that the viral proteins translated from genomes that initially enter the cell are able to induce vesicles in mouse cells. Therefore, interactions of wild-type viral proteins with membranes or with host cell proteins required for vesicle formation are presumably not compromised in mouse cells. The absence of HRV39 viral replication in mouse cells demonstrates that HRV39 must be blocked at a step subsequent to vesicle formation but prior to the initiation of replication, such as replication complex assembly or RNA synthesis. The replication complex is believed to form in cis
, coupling vesicle formation, genome translation, and viral RNA replication (67
). Preformed vesicles are unable to support replication of superinfecting poliovirus (27
), and trans
-complementation with nonstructural viral proteins is extremely limited (39
). Our data suggest that the vesicle-forming functions of the 2B and 3A proteins (or their precursors) are not linked to their ability to facilitate replication. The viral precursor proteins may recruit cellular proteins at a step prior to the formation of vesicles that either initiates or nucleates replication complex formation. Alternatively, replication complexes may form and subsequently require cell proteins to render them replication competent. As wild-type HRV39 is able to replicate efficiently in HeLa R19 cells, the block to HRV39 growth in mouse cells cannot be due to viral cis
-acting factors. Host cell proteins provided in trans
must play a role in the formation of a functional replication complex. Efforts to isolate these proteins are under way.