Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces

ABSTRACT

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed.

IMPORTANCE The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.

INTRODUCTION

Respiratory viruses are the leading cause of disease in humans (1). Their importance has been dramatically underscored by the consequences of the ongoing COVID-19 pandemic caused by the coronavirus SARS-CoV-2. In addition to coronaviruses, medically and socioeconomically important respiratory viruses include influenza virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, adenovirus, bocavirus, and respiratory enteroviruses. Human rhinovirus (HRV) (2–4), a respiratory enterovirus, is one of the most frequent human pathogens worldwide and is the major causative agent of common colds. HRV infections lead to huge economic losses every year (5). Moreover, HRVs are causal agents of severe diseases, such as pneumonia in the elderly and the immunocompromised. They also exacerbate other severe diseases, including asthma and chronic obstructive pulmonary disease (COPD) (2–4). In 2019 COPD which was the third leading cause of death worldwide, with 3.23 million deaths (WHO Global Health Estimates). The rising concern about the medical and socioeconomic consequences of HRV infections has elicited a renewed interest in the development of anti-HRV drugs. Several promising HRV capsid-binding compounds with antiviral activity were identified years ago (6, 7), but they failed to pass clinical trials. Other anti-enterovirus compounds have been identified in recent years (8–10), but no specific drugs against HRV (or nearly any other respiratory virus) have been approved so far.

Conversion into effective drugs of viral infection-inhibiting lead compounds identified via screening, as well as the process of antiviral drug design, rely on the acquisition of fundamental knowledge of the targeted viral elements and of their roles in the infectious cycle (11). Protein-protein interfaces (12) in virus capsids (13) are involved in virion morphogenesis, the preservation of virion integrity outside the cell, biologically relevant conformational rearrangements of the viral particles, and viral genome uncoating with or without capsid disassembly (14). Because of their several critical roles during viral infection and their stereochemical specificity, interfaces between capsid protein subunits have been recognized as promising virus-specific targets for the development of new antiviral drugs (6, 7, 15–22).

During the last decades, a great deal of knowledge has been obtained on the structural and molecular biology of HRV (23, 24). Similarly to other viruses of the Picornaviridae family, the HRV virion (25, 26) is formed by a single-stranded (ss) RNA genome enclosed in a structurally simple, icosahedral T = 1 (P = 3) capsid of ~30 nm in diameter. The capsid is built from 60 copies of each of three virus proteins with a β-barrel fold (VP1, VP2, VP3) and an internal, extended polypeptide (VP4), which may be considered to be a detached N-terminal extension of VP2 (Fig. 1).

FIG 1.

Structure of HRV-B14 and residues at the interpentamer interfaces. (A) High-resolution topographic model of the HRV-B14 virion obtained from the crystal structure (PDB: 4RHV) (26). The average virion diameter is ~30 nm. Some 5-, 3-, or 2-fold icosahedral axes of symmetry are indicated by a green pentagon, yellow asterisk, or orange rhombus, respectively. The virion surface is depth-cued, using colors from deep blue (lower radius) to yellow (higher radius). (B) Scheme of the icosahedral HRV capsid. A biological protomer made of VP1 (blue), VP2 (green), VP3 (red), and the internal polypeptide VP4 (not visible) is delimited by a cyan line. Two neighbor pentamers are delineated in violet, with the interface between them schematically defined by the straight line delimited by two yellow asterisks that correspond to two capsid 3-fold axes of symmetry. A capsid 2-fold axis of symmetry at the center of this interface is indicated by a black rhombus. A capsid 5-fold axis of symmetry is indicated by a small green pentagon. (C) Ribbon model of two neighbor pentameric subunits in the HRV-B14 virion. The amino acid residues in each pentamer that define the interpentamer interface (see panel B) are represented as purple (upper pentamer) or yellow (lower pentamer) space-filling models.

Knowledge regarding HRV morphogenesis is limited, but it is generally thought to proceed similarly to other enteroviruses, such as poliovirus, whose assembly mechanism has been thoroughly studied. In the generally accepted model of poliovirus (and other enteroviruses) morphogenesis (27, 28), 5 identical protomers, each formed by one copy of the capsid proteins VP0, VP3, and VP1, associate to form a pentamer; and 12 pentameric intermediates bind the viral ssRNA in a coassembly process that leads to an immature virion. During a subsequent virus maturation step, an autoproteolytic reaction cleaves VP0 to yield VP2 plus VP4. The HRV virion is a conformationally dynamic, metastable particle that is prone to undergo conformational changes in vivo upon interaction with cell receptors or acidification.

The uncoating of the HRV genome involves receptor-mediated and/or acid-induced conformational transitions of the virus particle these rearrangements that lead to the externalization of VP1 N-terminal segments as well as the release of VP4 and of the viral ssRNA via transient openings in the capsid (29–34). Recent evidence obtained with different enteroviruses (including poliovirus, enterovirus 71, and HRV) indicates that transient openings that are likely involved in genome uncoating occur around the centers of the interfaces between neighbor pentamers in the capsid, where the 2-fold axes of symmetry are located (35–38). Thus, transient restructuring of the pentamer-pentamer interfaces in the virion and changes to interpentamer interactions are required for RNA release. This relatively high propensity of the HRV virion to undergo conformational rearrangements can also lead to its inactivation outside the cell by mild acidification or by moderate heating.

The above-mentioned results have shown that the interfaces between pentamers in HRV (and other enteroviruses) are functionally important for infection because they play critical roles in virus assembly, conformational stability, and genome release. However, these interpentamer interfaces involve many amino acid residues; the specific structural elements and amino acid residues responsible for the biological roles of these extended capsid regions are largely unknown. Pioneering mutational studies of picornaviruses, especially poliovirus, revealed several capsid residues that are important for assembly, conformational stability, or dynamics (39), and these results were followed by further mutational studies of poliovirus and other picornaviruses (for a few recent examples see references [40–43]). Mutational studies on the stability or the dynamics of the HRV virion have focused mainly on the role of residues surrounding hydrophobic capsid cavities (44–47) but not interpentamer interfaces. To our knowledge, no systematic mutational studies of HRV or other picornavirus interpentamer interfaces have been performed so far (with the exception of a previous study by our group on foot-and-mouth disease, and only the effect on infectivity was analyzed in that study [48]). In the present study, we have performed a systematic mutational analysis to determine: (i) which particular locations and residues along the HRV capsid interpentamer interfaces are relevant for viral infection; (ii) the stage(s) in the viral cycle in which those interfacial residues are individually involved, including capsid and virion assembly and the conformational stability of the infectious virion when subjected to physical aggression via heating. The results provide a structure-based functional dissection of the interpentamer interfaces in the HRV virion. Implications for a better understanding of the relationships between HRV molecular structure and function and for the development of novel capsid interface-binding anti-HRV agents are discussed.

RESULTS

Structural analysis of the interpentamer interfaces in HRV.

HRV species B, serotype 14 (abbreviated HRV-B14 or HRV14) was chosen as a model because of the large amount of structural and functional knowledge on HRV that was previously obtained using this serotype. An operational definition of the interface between each pair of pentameric subunits in the HRV-B14 virion (Fig. 1) was established by identifying the capsid amino acid residues involved in interpentamer interactions in the refined crystal structure (PDB ID: 4RHV) (26) (Fig. 2; Fig. S1). Within the constraints imposed for accepting that two chemical groups interact with each other (see the legend for Fig. S1), the interpentamer interface in HRV-B14 contains 99 amino acid residues (per protomeric subunit). These residues participate in a total of 523 van der Waals (vdW) contacts (of which 217 are carbon-carbon [C-C] contacts), up to 86 hydrogen bonds (H-bonds), and 5 ion pairs with residues of the neighboring pentamer.

FIG 2.

Capsid polypeptide (VP2, VP3, VP1, and VP4) segments involved in the interpentamer interface. Blue represents amino acid residues that establish interpentamer interactions through their side chains (with or without additional interactions through the main chain). Red represents residues that interact through their main chains only. Black residues are not directly involved in interpentamer interactions.

Most (79%) of the interfacial residues establish pentamer-pentamer interactions through their side chains (either exclusively or together with the main chain). Of the 78 interfacial side chains (Fig. 2; Fig. S1), 35% are fundamentally apolar, and 65% are polar (M, Y, and W side chains can form hydrogen bonds, but they were considered fundamentally apolar because of the large volumes of their generally buried hydrophobic parts). Of the 51 polar side chains, 60% are neutral, 16% are acidic, and 24% are basic (Fig. S2).

Only 8 (10%) of the 78 interfacial side chains establish >5 C-C contacts, and define a few widely scattered hydrophobic spots (Fig. S1B). All but one of these side chains are either close to a 3-fold axis or halfway between the 3-fold and the 2-fold axis. Only one of them is fairly close to the 2-fold axis at the center of the interpentamer interface. In contrast to the few side chains that define sizable hydrophobic spots, nearly half of the interfacial side chains are involved in a large number of interpentamer H-bonds (42%) and/or a few ion pairs (8%). Most H-bonds (Fig. S1C) and all of the ion pairs (Fig. S1D) cluster within two large, symmetrically equivalent regions, each located halfway between a 3-fold axis at either end of the interface and the 2-fold axis at its center. Very few H-bonded or ion-paired side chains surround the 2-fold axis.

To sum up, the interpentamer interface in the HRV virion is fundamentally polar and includes a significant excess of positively charged residues over negatively charged residues, especially at the mildly acidic pH of the endosomes (+3 per protomer, +180 per virion). Few interfacial side chains are involved in substantial interpentamer hydrophobic interactions, whereas half of them are involved in many H-bonds and/or a few ion pairs. The region around each 2-fold axis appears to be conspicuously deficient in presumably energetic interpentamer interactions.

Selection of capsid residues for a functional dissection of the interpentamer interfaces in the HRV virion.

A thorough mutational analysis was performed by alanine scanning of many residues located at the interpentamer interfaces (as defined above). A mutation to alanine specifically removes the side chain of the targeted amino acid (beyond the Cβ) and the interactions it establishes without introducing any other interactions and with the lowest probability of substantially altering the main chain conformation. Of the 99 residues at the interpentamer interface in HRV-B14, 27 are not targets for alanine scanning because they interact with the neighbor pentamer through the main chain and/or Cβ only. 32 (close to half) of the remaining 72 interfacial residues were chosen as a representative sample for functional analysis (Table 1).

TABLE 1.

Amino acid residues at the interpentamer interfaces in the HRV-B14 virion chosen for mutational analysis

Mutationa	Interpentameric interactions removed by mutation to Alab
	Ion pair	H-bond	vdW
H1027A			16 (10)
V1031			7 (5)
I1033A			4 (3)
L1034A			5 (3)
E1052A		mc(G2019)	10 (4)
N2020A		sc(T3149)	5 (0)
T2024A		mc(V3152)	4 (2)
Q2026A		3mc(D3154, L3157, Q3158)	17 (5)
S2054A		mc(P2056)	1 (0)
D2057A			1 (0)
S2059A		sc(H2099)	3 (0)
R2062A		2mc(M3146, D3148)	14 (2)
D2067A	R3142	sc(N3077)	3 (0)
T2070A		2sc(Q3076)	1 (0)
V2091A			3 (3)
H2099A		sc(S2059)	4 (1)
Q2111A		sc(Q3192)	5 (1)
N2113A		2sc(S3124, T3193)	7 (0)
F2117A			10 (10)
T2241A		mc(Q3192)	4 (1)
T2243A		sc(Q3192)
N3077A		sc(D2067)	4 (0)
S3124A		sc(N2113), mc(A2114)
K3126A		sc(N2195)	9 (4)
R3142A	D2067	mc(L2066), sc(S2068)	5 (0)
R3143A	E2151	mc(D2088), 2sc(E2151)	12 (2)
M3146A			13 (7)
T3149A		sc(N2020)
H3150A			5 (1)
Q3192A		2sc(Q2111, T2243)	10 (3)
T3193A		sc(N2113)	3 (2)
I3196A			3 (3)

^aFor each substituted amino acid residue, the first digit indicates the protein (VP1, VP2 or VP3), and the last three digits indicate the amino acid position, according to the numbering used in the PDB 4RHV file for HRV-B14.

^bThe cutoff distances considered were: for ion pairs, 3.8 Å; for hydrogen bonds, 3.5 Å; for vdW contacts, 0.5 Å longer than the sum of the vdW radii of the two interacting atoms. The total number of vdW contacts and, in parentheses, the number of C-C contacts, are indicated.

The rationale for choosing this set of 32 residues was as follows: (i) prolines (4) were excluded because the results of their mutation to alanine may be more difficult to interpret, as large conformational changes could occur; (ii) amino acid locations along all of the interpentamer interfaces were sampled; (iii) different types of side chains (according to polarity and charge) were included in proportions that are comparable to those determined for the complete interface (Fig. S2), though a slight under-representation of apolar side chains was unavoidable due to the decision to exclude prolines; (iv) side chains involved in different types of interpentamer interactions were included in proportions that are comparable to those determined for the complete interface; (v) side chains involved in a few interpentamer vdW contacts only were somewhat under-represented, as it was guessed that they could be functionally less important than the side chains involved in more energetic interactions; however, a fair sample of 5 side chains involved in a few (1 to 5) non C-C vdW contacts only were included in the analysis; and (vi) importantly, the mutational analysis of this set of 32 residues sampled a vast majority of the total interpentamer interactions, largely because removing either side chain of an interacting pair eliminates all of its mutual interactions.

Molecular determinants of HRV infectivity located at the interpentamer interfaces.

The 32 interfacial amino acid residues chosen (Table 1) were individually replaced by alanine via the introduction of the appropriate mutation in the capsid protein-coding region (P1) of a HRV-B14 infectious cDNA clone. In each case, the complete P1 region was sequenced. For the nonmutated HRV control virus and a representative sample of the tested mutant viruses (N2020A, Q2026A, N2113A, S3124A), the entire viral genome was sequenced. For every mutant analyzed, the introduced mutation was actually present, and no accompanying mutations were found.

The infectivity of each of the 32 mutant viruses and of the nonmutated HRV control was analyzed using the same conditions, by transfecting host cells with equal amounts of viral RNAs transcribed from the corresponding infectious clones. Viral progeny infectious titers were determined at different times (24, 48, and 72 h) posttransfection (p.t.) in plaque assays. For each mutant, absolute infectious titers obtained in duplicate in one experiment were normalized relative to the infectious titer obtained for the control wild type (WT) virus in the same experiment. Titers from at least two independent experiments were averaged. The results are summarized in Table 2.

TABLE 2.

Normalized infectious titers for HRV-B14 mutants

Infectivity level/groupa	Mutant HRV	Relative infectious titerb
		24 h p.t.	48 h p.t.	72 h p.t.
	WT	1	1	1
V	V1031A	0.7 ± 0.3	1.2 ± 1.0	2.0 ± 3.1
	T2024A	0.6 ± 0.4	0.3 ± 0.4	0.9 ± 0.5
	S2059A	1.6 ± 1.5	1.6 ± 1.4	1.5 ± 0.7
	D2067A	0.4 ± 0.5	0.2 ± 0.2	0.2 ± 0.1
	T2070A	0.6 ± 0.5	0.9 ± 0.5	3.6 ± 2.9
	T2241A	0.3 ± 0.0	0.2 ± 0.1	0.6 ± 0.6
	M3146A	3.5 ± 2.3	2.8 ± 2.0	1.2 ± 0.8
	T3149A	0.3 ± 0.4	0.4 ± 0.3	2.8 ± 3.9
	I3196A	1.0 ± 0.0	1.4 ± 0.1	4.5 ± 0.1
IV	H1027A	0.1 ± 0.1	(7.1 ± 1.8) × 10−2	0.1 ± 0.1
	I1033A	(8.3 ± 3.4) × 10−3	(1.1 ± 0.1) × 10−2	(1.9 ± 1.2) × 10−2
	S2054A	(4.8 ± 4.2) × 10−2	(7.0 ± 3.6) × 10−2	0.5 ± 0.3
	H2099A	<(0.2 ± 0.4)	(5.1 ± 7.0) × 10−2	(9.6 ± 1.9) × 10−2
	K3126A	ND	(1.8 ± 0.8) × 10−2	5.0 ± 5.9
III	N2020A	<(1.5 ± 2.4) × 10−3	(1.7 ± 1.9) × 10−3	(6.0 ± 6.9) × 10−2
	D2057A	(9.6 ± 6.4) × 10−4	(3.5 ± 4.0) × 10−3	1.0 ± 0.9
	V2091A	(1.6 ± 1.6) × 10−4	(3.0 ± 2.0) × 10−3	0.8 ± 0.8
	N2113A	<(3.4 ± 5.6) × 10−3	(3.1 ± 0.2) × 10−3	(5.1 ± 0.7) × 10−2
	T2243A	(2.7 ± 1.0) × 10−4	(6.5 ± 7.2) × 10−3	5.2 ± 8.7
	N3077A	(8.4 ± 6.2) × 10−4	(1.1 ± 0.9) × 10−3	1.0 ± 1.0
	R3143A	<(2.8 ± 4.3) × 10−4	(1.5 ± 0.1) × 10−3	0.5 ± 0.4
	H3150A	(1.2 ± 1.6) × 10−3	(1.7 ± 1.9) × 10−3	0.9 ± 0.2
II	E1052A	<(5.3 ± 6.1) × 10−4	(2.1 ± 3.2) × 10−4	(1.4 ± 1.9) × 10−2
	Q2111A	<(1.0 ± 1.2) × 10−4	(1.3 ± 0.9) × 10−4	0.3 ± 0.4
I	L1034A	<(1.8 ± 0.3) × 10−5	<(1.2 ± 0.3) × 10−5	<(1.3 ± 0.5) × 10−5
	Q2026A	<(5.1 ± 6.3) × 10−4	<(6.4 ± 5.9) × 10−6	<(3.7 ± 5.1) × 10−6
	R2062A	<(7.2 ± 6.0) × 10−5	<(1.1 ± 1.2) × 10−5	(8.5 ± 2.9) × 10−3
	F2117A	<(1.7 ± 0.4) × 10−4	<(1.5 ± 0.3) × 10−5	<(5.1 ± 4.8) × 10−6
	S3124A	<(9.6 ± 6.0) × 10−4	<7.2 × 10−7	(3.6 ± 4.0) × 10−5
	R3142A	<(1.0 ± 0.9) × 10−4	<(1.0 ± 0.9) × 10−4	0.1 ± 0.2
	Q3192A	<(1.7 ± 0.9) × 10−4	2.2 × 10−6	(7.1 ± 4.0) × 10−4
	T3193A	<(7.5 ± 9.4) × 10−5	<7.2 × 10−5	8.2 × 10−7

^aThe mutant viruses were classified into 5 levels/groups according to the infectious titer (relative to the WT virus control) of the viral progeny at 48 h p.t. (immediately after a complete cytopathic effect was observed for the WT virus control). Group V, relative infectious titer between 1 and 10⁻¹ (similar to the WT); group IV, relative titer between 10⁻¹ and 10⁻²; group III, relative titer between 10⁻² and 10⁻³; group II, relative titer between 10⁻³ and 10⁻⁴; group I, relative infectious titer of <10⁻⁴ (undetectable).

^bNormalized infectious titers (average ± standard deviation) of the viral populations obtained at 24 h, 48 h, or 72 h p.t., relative to the titer of the WT HRV-B14 control. The absolute titers obtained for the HRV-B14 wt virus (average ± standard deviation) were (1.8 ± 1.8) × 10⁶ PFU/mL at 24 h p.t., (1.1 ± 1.6) × 10⁷ PFU/mL at 48 h p.t., and (4.4 ± 0.1) × 10⁷ PFU/mL at 72 h p.t. ND, not determined.

The 32 virus mutants tested were classified into 5 infectivity levels (groups I to V), according to progeny virus titers determined at 48 h p.t. (after a complete cytopathic effect was observed for the WT control). This grouping separates mutants whose infectivity was 1 order of magnitude lower than that of the previous category, from group V (titer similar to the WT) to group I (titers less than 10⁻⁴ of the WT) (Table 2). This grouping in 5 infectivity levels had already been used for a different picornavirus in a previous study with a different goal (48). In the present study, this categorization facilitated the distinction between different viral phenotypes associated with clearly different infectivity levels as well as the comparison of evolutionary conservation versus biological relevance (see below). Only 28% of the mutations had either relatively little or no significant effect on infectivity (relative titer >10⁻¹, group V); 16% had a substantial effect (relative titer between 10⁻² and 10⁻¹, group IV); 25% had a severe effect (relative titer between 10⁻³ and 10⁻², group III); 6% had a drastic effect on infectivity (relative titer between 10⁻⁴ and 10⁻³, group II); and 25% of the mutations led to undetectable infectious titers (group I).

It was important to ascertain whether the negative effects of most mutations on viral infectivity were directly due to the mutations introduced and not to other mutations that could have been fixed in the viral population during multiplication in the host cells. It was also important to confirm that the close to normal infectivity of some tested mutants was not due to genotypic reversion or to the fixation of compensatory secondary mutations. Thus, the viral RNA present in cell culture supernatants obtained 48 h after transfection with each mutant or with the control WT virus was reverse transcribed to DNA and amplified by PCR, and the entire capsid-coding region was sequenced. No viral RNA could be recovered from many of the group I (lethal) mutants, as expected, but for 27 other mutants, the amount of viral RNA was high enough to allow amplification and sequencing (Table S1). In every case, the consensus sequence of the progeny population confirmed that the introduced mutation was still present. In the vast majority of these mutants (23 out of 27) and in the WT control, no accompanying mutations leading to amino acid substitution were either detected or present in a significant proportion (>25%); in 4 mutants only, 1 or 2 accompanying mutations were present in a sizable proportion (26% to 56%), but they were far from fully imposed (Table S1).

When viral progeny titers were determined at longer times p.t. (72 h instead of 48 h), it was observed that 11 out of 23 mutants of groups I to IV yielded close to normal viral progeny titers (Table 2). To determine whether other mutations were imposed late in the progeny virus population, the viral RNA present in cell supernatants at 72 h p.t. was amplified, and the complete capsid-coding region was sequenced (Table S1). In one mutant only (R3142A, group I), the original mutation had reverted to WT, explaining its dramatic recovery of infectivity. For the remaining mutants and the WT control, no reversion and no additional mutations leading to amino acid substitution were either detected or present in a significant proportion. The close to normal mutant virus yields at longer times appears to be simply due to the infection of nontransfected cells by the reduced number of infectious progeny viruses initially produced in the fraction of cells that were successfully transfected.

To sum up, for (nearly) all of the virus mutants whose infectivity was abolished or reduced, the observed deleterious effect can be interpreted as a direct consequence of the truncation of a single amino acid side chain at the interpentamer interface. One quarter of the tested interfacial side chains are critical determinants of infection (i.e., the removal of any of them was lethal for the virus), and as many as three quarters of them have an important role in HRV infection. These results reveal the importance of most molecular components and interactions at the capsid interpentamer interfaces for rhinovirus infection.

Chemical features and distribution at the HRV interpentamer interfaces of functionally relevant residues.

Next, we analyzed the chemical features, type, and number of interactions, as well as the particular locations at the interpentamer interfaces, of different residues that were found to contribute to virus infectivity. No particular chemical features (side chain type) or numbers and types of interactions could be specifically associated with biological function. The location of each of the 32 residues analyzed at the interpentamer interface is shown in Fig. 3, color-coded according to the infectivity group to which it was ascribed. The analysis revealed that: (i) residues critically relevant for infectivity (group I, colored red in Fig. 3) are rather uniformly scattered along most of the interpentamer interface; (ii) however, the small area that surrounds the interface center (capsid 2-fold axis) is conspicuously free from those critical residues; and (iii) interfacial residues with important (though not as critical) roles in infectivity (groups II, III, IV, respectively colored orange, green, or cyan in Fig. 3) appear to be randomly scattered along all of the interpentamer interface. Thus, no sizable region within the long interpentamer interfaces in HRV is devoid of biological function, and no sizable region at these interfaces in HRV appears to be biologically more relevant than the others.

FIG 3.

Distribution of amino acid residues at the interpentamer interfaces of HRV-B14, according to their effect on virus infectivity (at 48 h p.t.). The structure that surrounds the interface between two neighbor pentamers in the HRV-B14 virion is represented as a ribbon model. In each panel, the interpentamer interface runs horizontally from one capsid 3-fold axis located at the left side of the image to another 3-fold axis at the right side of the image (compare to Fig. 1C). Residues contained in the interpentamer interfaces are represented as space-filling models and are color-coded: red, lethal effect on infectivity (group I); orange, drastic effect (group II); green, severe effect (group III); cyan, moderate effect (group IV); dark blue, no substantial effect (group V).

Evolutionary conservation in HRV of residues at the interpentamer interfaces.

It was then analyzed whether the high density of infectivity-determining residues at the interpentamer interfaces in HRV was reflected in a high conservation of interfacial residues among HRV isolates and species. The capsid sequences of 123 HRV strains from different serotypes and species (A, B, or C) were aligned, a consensus sequence was determined, and the percent conservation was determined for each capsid residue. When the complete capsid was considered, 78% of the 915 residues per protomer showed a 60% or higher degree of conservation, and 37% were fully (100%) conserved (Fig. 4). The distribution of residues with different degrees of conservation was, however, far from uniform. For example, as a whole, the capsid outer surface contained many nonconserved residues and only a few highly conserved residues, whereas the capsid inner wall included a high proportion of highly conserved residues (Fig. 4A). Remarkably, the interpentamer interface was one of the most conserved regions in the HRV capsid (Fig. 4B). 91% of the 99 residues per protomer that are involved in interpentamer interactions showed a 60% or higher degree of conservation, and 50% were fully conserved.

FIG 4.

Degree of conservation of capsid amino acid residues in 123 HRV strains from different serotypes and species (HRV-A, -B, or -C). A capsid pentamer is depicted. The accompanying inset show a side view of the same pentamer. The percent conservation for each amino acid residue is color-coded from deep blue (100% conservation) to deep red (15% conservation). (A) Degree of conservation of every capsid residue. Left image, top view; right image, bottom view. (B) Degree of conservation of the residues located at pentamer-pentamer interfaces. Left image, top view; right image, bottom view.

It could be expected that the biologically most critical residues at the interpentamer interfaces would be among the most conserved in the HRV capsid. Accordingly, all but one of the 10 tested residues from infectivity groups I or II (abolished or dramatically reduced infectivity) were fully conserved (8 residues) or nearly fully (99%) conserved (one residue). The only exception, residue 3124, was also 99% conserved if no distinction is made between serine and threonine, which are chemically similar and differ by size in just one methyl group (Fig. S3). In contrast, the 22 tested residues from infectivity groups III, IV, or V (less drastic to insignificant infectivity reductions) were much less conserved, with an average conservation that was not significantly different from the capsid average (about 75%, 74%, or 79% for groups III, IV, or V, respectively). Only 8 of those 22 residues (36%) were 99 to 100% conserved, again similar to the capsid average (Fig. S3).

Interestingly, some of the 100% conserved residues could be mutated to alanine without a significant reduction in virus infectivity (e.g., S2059, M3146, group V) (Fig. S3). This observation suggests that, even though these residues are not required for infection of cultured cells, they may be important for maximum biological fitness and/or for additional viral functions required for the productive infection of humans. Thus, the proportion of biologically critical residues at the interpentamer interfaces for the in vivo propagation of HRV is probably even higher than that found in this study.

Conversely, some nonconserved residues could not be mutated to alanine without substantial to severe reductions of infectivity. For some of these cases, the explanation could not be simply based on a presumed intolerance to alanine in that position. For example, residues 1033 (group IV) or 3077 (group III) were alanine in 60% or 45%, respectively, of the 123 infectious HRV isolates compared (Fig. S3). The latter result provides evidence that functionally compensatory mutations may be occurring at the interpentamer interfaces in HRV. Additional support for this possibility comes from direct evidence for frequent compensatory mutations found at interpentamer interfaces in another picornavirus, foot-and-mouth disease virus (49, 50).

Effects of biologically relevant residues at the capsid interpentamer interfaces on HRV assembly.

A possibility to explain the role on HRV infection of many different amino acid side chains at the capsid interpentamer interfaces is that they are important for virus assembly through the intermolecular interactions they establish. To analyze the effects of interfacial mutations on virus morphogenesis, mutant HRV RNAs, and also the nonmutated HRV-B14 RNA used as a control, were transcribed in vitro from the corresponding cDNA clones and used to transfect host cells under the same conditions. This time, the transfection conditions were adjusted to achieve viral RNA uptake by most (close to 80%) of the cells (see Materials and Methods). In this way, viral particles were produced in essentially one step only, avoiding significant reinfection by any infectious virions emerging from the originally transfected cells. Viral particles produced in a single infectious cycle in the transfected cells were detected via radioactive (³⁵S-Met/Cys) metabolic labeling and ultracentrifugation analysis using density gradients. For these assays a fair, representative sample of 12 mutant HRVs belonging to the 5 different infectivity groups was selected. These 12 residues included about half of the residues from each infectivity group from IV to I (i.e., of all the mutants that showed reduced infectivity by different orders of magnitude). In addition, for each infectivity group, mutations that involved either polar or apolar residues involved in different types of noncovalent interactions (e.g., electrostatic interactions versus hydrophobic contacts) were chosen. The mutants from group V had no significant effect on infectivity. Thus, only 2 mutants out of 9 from group V were included to show that they did preserve assembly as expected.

For each mutant HRV tested, relative amounts of full virions (sedimentation coefficient 150S) and empty capsids (80S) were obtained by integrating the corresponding peaks in the gradient and by normalizing the value to that obtained for the nonmutated HRV control in the same experiment (see Materials and Methods). Transfection bypassed most steps in the infectious cycle where viral particles are involved, including virus binding to the cell receptor, entry into the cell, intracellular transport, and viral genome uncoating. In addition, it was previously shown that the deletion of the capsid coding sequence of HRV-B14 had no effect on viral genome replication, with the exception of a small (33 nucleotide long) sequence (cre element) (51, 52). As only single point mutations in the coding sequences of the capsid protein genes were tested in this study and none of them were contained within the cre element, any reduction in infectivity could not be ascribed to viral genome replication. Densitometry of capsid protein bands in well-resolved electrophoresis gels of transfected cell extracts revealed that none of the 12 mutations tested had any significant effect on the amount of viral capsid protein produced (Fig. S4). Thus, any reduction in the amount of virions produced could likely be due to a defect during virus morphogenesis. The mutations tested involve the substitution of single amino acids, every one of them located on the surface of the free capsid protein and at the interpentamer interfaces in the assembled viral particle. Thus, a reduction in the amount of virions produced would point to an impaired association between pentamers, though an effect on a previous morphogenetic step cannot be completely excluded.

The results of a representative experiment are shown in Fig. 5A. The relative viral particle production efficiencies obtained for the complete set of mutant HRVs are given in Fig. 5B. As expected, the 2 representative mutants from group V (infectivity similar to that of nonmutated HRV) yielded virion production efficiencies that were comparable (same order of magnitude) to the HRV-B14 control (relative efficiency of 17% or 34% of that of the control). In contrast, 9 out of 10 representative mutants from groups IV, III, II, or I (infectivity substantially or drastically reduced) showed virion production efficiencies that were drastically reduced (relative efficiency of 5% of that of the control for 2 mutants and <1% for the other 7 mutants) (Fig. 5B). Moreover, a qualitative relationship was observed between reduced virion production and reduced virus infectivity.

FIG 5.

Effect on the HRV assembly efficiency of mutations at the capsid interpentamer interfaces. (A) The results of a representative experiment are shown. The plot represents ultracentrifugation profiles (radioactivity versus gradient fraction number) obtained for radiolabeled viral particles produced in transfected host cells. Markers corresponding to the sedimentation coefficients of 80S (empty capsids) or 150S (full virions) are indicated by arrows. Black, negative-control corresponding to a lysate from mock-transfected cells; red, positive-control corresponding to nonmutated HRV-B14; blue, H1027A mutant; yellow, D2057A mutant; green, Q2111A mutant. (B) Relative assembly efficiency values obtained for every mutant tested. Mutants are classified according to their infectivity level, from group V (infectivity similar to that of the WT) to group I (undetectable infectivity) (see Table 2). For each mutant, the numbers indicate (as percent values) the estimated amounts of virions and empty capsids relative to the amount of WT virions. To facilitate comparison, in the virion (150S) row, black, gray, or white squares, respectively, indicate that the amount of assembled virions was similar to the amount of WT virions (>15%), was substantially lower than the amount of WT virions (between 5% and 15%), or was undetectable (<5%). In the empty capsid (80S) row, white, gray, or black squares, respectively, indicate that the amount of empty capsids was undetectable (<5% of the amount of WT virions), low (between 5% and 15% of the amount of WT virions), or substantial (>15% of the amount of WT virions).

Remarkably, for 5 out of the 9 mutants for which no or drastically reduced amounts of virions were formed, significant amounts of 80S empty capsids were detected (up to 25% of the amount of virions obtained with the nonmutated HRV control) (Fig. 5B). Electron microscopy showed that the 80S mutant empty capsids had the right size (30 nm diameter), spherical shape, and aspect after staining (Fig. 5A). Half of the 8 tested mutants with no or highly reduced infectivity (groups I, II, and III) yielded substantial amounts of empty capsids (10 to 25%), while the 2 tested mutants with a less drastic reduction of infectivity (group IV) yielded lower amounts of empty capsids (1 to 8%). Enterovirus morphogenesis appears to require the coassembly of capsid pentameric intermediates and the viral RNA (27, 28). The possibility that single amino acid substitutions at the interpentamer interfaces would make the presence of the viral RNA dispensable for the assembly of HRV particles seems highly unlikely. Thus, the presence of empty capsids may indicate that some mutant virions were indeed assembled but were conformationally labile enough (through weakened pentamer-pentamer associations) to allow for the easy escape of the viral RNA, which led to a loss of infectivity and left an empty capsid as the observed end product. Alternatively, if HRV morphogenesis would proceed through an empty capsid intermediate, the sizable amounts of empty capsids detected with some mutants could indicate that the corresponding mutations impair RNA packaging into the preformed capsid. Whatever the case, virus capsid assembly was indeed impaired for those mutants, because the sum of the amount of virions (if any) plus the amount of empty capsids was much less than the amount of nonmutated virions produced in the same experiment.

From an estimation of viral particles by electron microscopy and infectious units in the same preparation via titration on plaque assays, the particle to plaque-forming unit (PFU) ratio for the wild type HRV virus control was of the order of 1000:1. For each mutant, the variation in the particle to PFU ratio relative to the wild type value was obtained by estimating the relative amount of full plus empty viral particles (the sum of the areas under the 150S and 80S peaks) divided by the relative infectious titer (Fig. S5). For all but one of the group V, IV, or III mutants, the particle to PFU ratio was similar to the WT particle to PFU ratio. The exception was mutant D2057A, for which the particle to PFU ratio increased by 70-fold, relative to the WT. For the group II and I (lethal) mutants tested (Q2111A, L1034A, F2117A, S3124A), the particle to PFU ratios increased by over 1000-fold, relative to the WT. These high ratios were specifically due to the fact that a sizable number of (noninfectious) empty capsids were detected against low numbers of infectious virions. These results are consistent with the reduction in infectivity by most group IV or III (low infectivity) mutants being largely due to impaired assembly, whereas the reduction in the infectivity of most group II or I (extremely low infectivity or lethal) mutants could be the result of a combination of impaired assembly, reduced stability and, perhaps, unproductive RNA release.

Effects of biologically relevant residues at the capsid interpentamer interfaces on HRV stability.

Capsid residues involved in pentamer-pentamer interactions could have a role not only in virion assembly but also in preserving the infectious conformation of the virion if subjected to physical aggression (e.g., heat or pH extremes) out of the cell. The thermostability of a representative sample of 12 HRV mutants for which infectious virions could be obtained (from groups II, III, IV, or V, all except group I, which is composed of lethal mutants) was determined and compared to that of nonmutated HRV-B14. The same subset of 12 mutants that had been tested for virion assembly was considered for assaying virion stability. However, as the mutants from group I are lethal, no testing of its stability was possible. This left only 9 mutants out of the 12 mutants assayed for assembly to be tested for stability. Thus, we decided to increase the sample size for testing stability by including 4 more mutants that showed different reductions in infectivity (2, 1, and 1 more mutants from groups IV, III, and II, respectively), even though these 4 mutants had not been tested for assembly. In each experiment, the same amount of each mutant virion and of the nonmutated virion as an internal control were heated at 45°C for either 45 min or 120 min, and the remaining infectious virions were determined via titration in plaque assays using cultured cells.

The results of a representative experiment are shown in Fig. 6A. The relative thermostability values obtained for the complete set of mutant HRVs after incubation for 45 min at 45°C are indicated in Fig. 6B. As expected, the tested representative mutant from group V (infectivity similar to that of nonmutated HRV) was as stable as the nonmutated HRV (relative stability of 104% of that of the control). In contrast, all four representative mutants from group IV (infectivity substantially reduced) were substantially or dramatically destabilized (relative stabilities of 35%, 21%, 0.4%, or 0.4% of that of the control). Remarkably, 6 of the 7 representative mutants from groups III or II (infectivity drastically reduced) were only moderately destabilized (relative stabilities of 55% or 37%) or were not significantly destabilized (relative stabilities between 112% and 70%) (Fig. 6B). Results obtained after heating for 120 min confirmed the destabilization observed for the mutants when they were heated for 45 min only.

FIG 6.

Effect on HRV14 virion thermostability (resistance to thermal inactivation of infectivity) of mutations at the capsid interpentamer interfaces. (A) The results of a representative experiment are shown. The bar plot represents the reductions in the normalized virus titers after incubation for 45 min or 120 min at 45°C. Black bars: WT HRV-B14 used in each experiment as an internal control; gray bars, mutant H2099A; white bars, mutant E1052A. Error bars are indicated. (B) Relative thermostability values obtained for every mutant tested. The mutants are classified according to infectivity level, from group V (infectivity similar to that of the WT) to group I (undetectable infectivity) (see Table 2). The bars represent the percent difference in thermostability between each mutant and the nonmutated HRV-B14 control after incubation for 45 min at 45°C. The values are the averages of two or four duplicate measurements in one or two independent experiments.

The above results revealed that about half of the tested amino acid residues at the interpentamer interfaces, in addition to being involved in virion morphogenesis, are also involved in the conformational stabilization of the infectious virion. Destabilization did not show a correlation with assembly defects or reduced progeny yields.

Distribution at the HRV interpentamer interfaces of residues involved in virion assembly or conformational stability.

The location of each of the many residues at the interpentamer interface that were analyzed for their roles in virion assembly or conformational stability is shown in Fig. 7A or Fig. 7B, respectively. The residues critically relevant for assembly, as well as those that were also important to prevent the release (or encapsidation) of the RNA (colored red or yellow, respectively), are rather uniformly scattered along most of the interpentamer interface (Fig. 7A). Interestingly, every tested residue around the capsid 3-fold axes were critical for virion assembly, whereas a large fraction of the residues at the capsid 2-fold axis (in the center of each pentamer-pentamer interface) were dispensable for virion assembly. The residues critically relevant for stability (colored red) are again rather uniformly scattered along most of the interpentamer interface (Fig. 7B). To sum up, the above results indicate that nearly all of the tested amino acid residues at the interpentamer interfaces, irrespective of the residue type, the interactions established, or the location at the interface, have a major role during HRV infection because they are critically involved in virion morphogenesis and/or conformational stability.

FIG 7.

Distribution of amino acid residues at the interpentamer interfaces of HRV-B14, according to their roles in virion assembly (A) or virion stability against thermal inactivation (B). The structure that surrounds the interface between two neighbor pentamers in the HRV-B14 virion is represented as a ribbon model. In each panel, the interpentamer interface runs horizontally from one capsid 3-fold axis located at the left side of the image to another 3-fold axis at the right side of the image (compare to Fig. 1C). Residues contained in the interpentamer interfaces are represented as space-filling models and are color-coded. In (A), colors represent their effect on virion assembly: red, critical for virion assembly (>85% reduction in the amount of assembled virions when the residue was mutated to alanine); yellow: critical for both virion assembly and for preventing the appearance of empty capsids; green: insignificant effect on virion assembly (<85% reduction in the amount of assembled virions when the residue was mutated to alanine) (see Fig. 4B). In (B), colors represent their roles on virion thermostability: red, major role (>80% destabilization when the residue was mutated to alanine); yellow, substantial role (>60% destabilization on the mutation to alanine); green, no significant role (no significant destabilization when the residue was mutated to alanine) (see Fig. 5B).

DISCUSSION

The present structure-function dissection of the interfaces between pentameric subunits in the HRV virion reveals a rather unusual type of protein-protein interface. Interfaces in many cellular protein oligomers or assemblies are made of a central core of hydrophobic residues surrounded by a rim of polar residues. These interfaces frequently contain a few “hot spots” formed by hydrophobic residues that provide most of the binding energy (12). Some viral capsid interfaces also share those features. They include the interface between the C-terminal domains of neighbor capsid protein CA subunits in the mature immunodeficiency virus capsid (53). In addition, major molecular determinants for capsid assembly at the extended interface between VP2 subunits in the parvovirus minute virus of mice (like HRV, a nonenveloped icosahedral T = 1 virus) were traced to isolated, regularly spaced patches of few residues involved in many hydrophobic contacts or buried hydrogen bonds and/or salt bridges (54). In contrast to those viral capsid interfaces, in the interpentamer interface of HRV the hydrophobic residues are scarce and randomly scattered, and they form no conspicuous hot spots. The capsid pentamers bind to each other mainly through relatively weak but abundant electrostatic interactions (van der Waals, hydrogen bonds, some ion pairs) between polar residues scattered along the interface. The results indicate that the vast majority of the interfacial capsid residues are important for HRV infectivity because they participate in many weak, polar interactions between pentamers.

An adequate compromise between conformational stability and instability of the virion has been proven to be essential for maximizing the infectivity of several viruses (14). The results of the present study indicate that, for HRV, many residues at the pentamer-pentamer interfaces are important for infectivity, largely because they enable capsid assembly. Moreover, about half of the tested residues are also important for conferring adequate resistance of the virion to unproductive conformational changes or disintegration.

Moreover, the observation of empty capsids as the final product of transfection for a sizable number of the mutant viruses analyzed here suggests that a fair proportion of the interfacial residues may also be involved in other processes. Abundant evidence favors the possibility that enterovirus morphogenesis proceeds through capsid-RNA coassembly (27, 42) (see the Introduction). If such were the case for HRV, the interfacial residues whose mutations led to substantial amounts of empty capsids (S2054, T2243, Q1111, L1034, F2117) could have a role in modulating a conformational transition that leads to the release of RNA from virions. During HRV genome uncoating, the viral RNA exits the capsid through transient openings formed close to the 2-fold axis at the center of each interpentamer interface. None of the tested mutant viruses that included amino acid substitutions closer to the 2-fold axis belonged to group I (no detectable infectivity). However, most of these mutations around the 2-fold axis (E1052A, D2057A, S2054A, H2099A) led to capsids that were devoid of RNA and/or facilitated the dissociation of the viral capsid into subunits. It is tempting to propose, as a tentative model to be tested, that these residues could keep the 2-fold axis regions in a closed state under normal conditions, thereby preventing the untimely release of RNA but allowing productive uncoating under the appropriate stimuli, such as receptor binding or acidification.

An alternative model for HRV morphogenesis, in which an empty capsid intermediate is assembled first in the absence of viral RNA, followed by RNA packaging into the preformed capsid, cannot be excluded. If such were the case, the interfacial residues whose mutation led to the detection of sizable amounts of empty capsids could have also a role in enabling RNA packaging into a preassembled capsid. Further studies are required to definitively establish whether HRV morphogenesis proceeds via capsid-RNA coassembly or via RNA packaging into a preformed capsid.

From an applied perspective, the results suggest that, for the design of a drug that could inhibit HRV assembly or stability, the interpentamer interface constitutes an excellent target. First, the rational design of interface-binding compounds with anti-HRV activity may not require the identification of any functional hot spot. In fact, biologically critical residues and elements were found to be distributed at a high density along most of the interface and were not clustered at any specific site. Moreover, most interfacial residues were evolutionarily more conserved than those in many other capsid regions. This finding suggests that the virus may have more difficulties in escaping from an interface-binding drug through mutation without losing biological fitness and fewer chances to recover fitness via the fixation of secondary mutations (49, 50). It must be noted, however, that some other interfacial residues involved in viral infection were much less conserved, presumably due to the occurrence of compensatory mutations. Thus, the design of an interface-binding compound should focus on the creation of specific interactions with a set of the many biologically important interfacial residues that are conserved. To sum up, there is a possibility that any small organic compound designed for high-affinity binding to any specific cavity or localized structural element at the interpentamer interface will inhibit HRV infection because any of those sites may be important for virion assembly and/or stability. If the compound is designed to bind some subset of the many conserved interfacial residues, it may prove effective for different HRV variants, and the probability of emerging drug-escape mutants may be reduced.

Conclusion.

The image that arises from a thorough structure-function dissection of the extended, elongated interfaces between capsid subunits in HRV, unlike those of other viruses analyzed, is not one of conserved functional hot spots and structurally compartmentalized functions. Instead, these large interfaces constitute a quasi-continuum of biologically relevant residues. Most of them are critically involved in virion assembly, and many of them are also involved in preserving an adequate conformational stability and/or resistance to the disintegration of the virion under thermal stress.

MATERIALS AND METHODS

Recombinant plasmids and site-directed mutagenesis.

The recombinant clone pWR3.26, containing the complete sequence of the HRV-B14 viral genome, was obtained from the American Type Culture Collection (ATCC), amplified via the transformation of E. coli XL1-Blue, and purified (55). Mutations were introduced in the HRV-B14 capsid-coding region contained in pWR3.26 using the QuikChange II XL Site-directed Mutagenesis Kit (Stratagene) and following the procedure described by the manufacturer. The presence of the desired mutation and the absence of secondary mutations was determined by automated DNA sequencing.

In vitro transcription of the HRV genome.

pWR3.26 plasmids containing the desired mutations and the WT control were linearized with restriction endonuclease MluI and purified via phenol extraction. RNA from the WT or mutant HRV-B14 were transcribed from 1 μg of purified and linearized DNA using 20 U of T7 RNA polymerase (Promega) in transcription buffer (50 mM Tris-HCl [pH = 7.9], 12.5 mM NaCl, 7.5 mM MgCl₂, 2.5 mM spermidine, 1.25% BSA, 10 mM DTT, 40 U RNasin, and 1 mM of each ribonucleoside triphosphate). After incubation of the mixture for 2 h at 37°C, the transcribed RNA was stored at −80°C for a short time until use. Both linearized plasmid DNAs and transcribed infectious viral RNAs were quantified by agarose gel electrophoresis and spectrophotometry using a NanoDrop 1000 apparatus (Thermo Scientific).

Transfection of host cells with HRV genomic RNA.

HeLa-H1 cells were obtained from the ATCC. Cells were transfected by electroporation with nonmutated or mutant HRV-B14 viral RNA transcribed from the corresponding pWR3.26 recombinant plasmids. For comparing progeny virus production in quantitative assays, the same amount (15 μg in PBS) of WT and mutant viral RNAs was used for the electroporation of the same number of cells under the same conditions. The electroporation of 4 × 10⁶ cells was performed in a 0.5 mL cuvette with one pulse at 980 V, 25 μF, and maximum resistance in a Gene Pulser II apparatus (Bio-Rad). Control cells were electroporated using the corresponding volume of PBS instead of a RNA solution. Transfected cells were grown at 35°C in petri dishes (60 mm in diameter) for 4 h with 4 mL of Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM glutamine and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) (termed DMEMc) and supplemented with 10% fetal bovine serum (FBS). This was followed by prolonged incubation with the same medium supplemented with 2% FBS. Supernatants from transfected cell cultures were collected at 24 h, 48 h, and/or 72 h p.t., depending on the experiment. Transfection efficiency was determined by counting the number of transfected cells as infectious units and dividing it by the total number of cells.

Determination of HRV infectious titers.

Virus suspensions obtained from transfected cells were titrated in standard plaque-formation assays. Nearly confluent HeLa-H1 cell monolayers in 60 mm-diameter petri dishes were inoculated with 200 μL of serial dilutions of a virus suspension in PBS with 0.1% BSA. Virus adsorption was left to proceed for 1 h at 35°C. The inoculum was then removed, and the cells were overlaid with a solid layer of 0.7% agar in DMEM containing 1% FBS and 0.5% DEAE Dextran. After incubation at 35°C for 72 h, the cells were fixed with formaldehyde and stained with crystal violet. To ensure comparable results, a nonmutated virus was included in each experiment as a control. Titers were obtained at least in duplicate in each experiment, and the mean relative titers obtained for each mutant in independent experiments were averaged.

Extraction of RNA from HRV viral populations.

The viral RNA present at 48 or 72 h p.t. in supernatants from the WT or mutant HRV-B14-transfected cell cultures were obtained by mixing 150 μL of the supernatant with 400 μL of TRIzol LS (Invitrogen). After shaking, the mixture was incubated for 5 min at room temperature, 200 μL of chloroform were added, and the mixture was shaken, incubated for 10 min at room temperature, and centrifuged in a microcentrifuge at 14,000 rpm for 15 min at 4°C. The aqueous phase was collected and 250 μL of 2-propanol were added to precipitate the RNA for 2 h at −20°C. The precipitate was collected by centrifugation at 14,000 rpm for 10 min at 4°C, washed with 0.5 mL of 70% ethanol, recentrifuged, resuspended in 30 μL of sterile water containing 0.1% diethylpyrocarbonate (Sigma), and briefly stored at −80°C.

Reverse transcription of RNA from HRV, DNA amplification, and sequencing.

The desired coding regions from the HRV-B14 RNA obtained from transfected cells was reverse-transcribed to cDNA and amplified via RT-PCR using specific oligonucleotide primers. The reaction mixture (50 μL) contained 1 to 2 μg of RNA, 100 ng of each oligonucleotide primer, 5 μL of 10× reaction buffer 3 (Roche), 1.5 mM MgCl₂, 0.2 mM of each deoxyrribonucleoside triphosphate (Invitrogen), 0.3 μL of RNasin (40 U/μL, Promega), 1 μL of reverse transcriptase (AMV-RT, 10 U/μL, Roche) and 1 μL of thermostable DNA polymerase (EHF, 3.5 U/μL, Roche). The reactions were carried out under the following conditions: reverse transcription, 1 h at 42°C; initial denaturation, 2 min at 95°C; 30 cycles of denaturation (30 s at 95°C), annealing (60 s at 55°C or 60°C), and extension (210 s at 68°C); and final extension, 10 min at 72°C. After the reactions were complete, the mixture was briefly stored at 4°C until use.

The products of the RT-PCR were analyzed via agarose gel electrophoresis and purified using the Wizard SV Gel and PCR Clean-Up System (Promega), following the manufacturer’s instructions. The purified viral DNA was quantified via spectrophotometry, and the HRV-B14 capsid-coding region was sequenced as indicated above.

Virion assembly assays.

HeLa-H1 cells were transfected with nonmutated or mutated viral RNA as described above and metabolically radiolabeled as described previously (46, 56). Briefly, transfected cells in 60 mm-diameter petri dishes were incubated for 1 h at 35°C in 4 mL DMEMc supplemented with 10% FBS. Then, the cells were washed 5 times with PBS and incubated for 2 h at 35°C in DMEMc supplemented with 5% FBS. After 5 washes with cystine and methionine-free DMEMc, the cells were grown for 8 h at 35°C in 2 mL of cystine and methionine-free DMEMc containing 200 μCi methionine/cysteine [³⁵S] (PerkinElmer) and supplemented with 5% FBS. The monolayers were then washed 5 times with PBS and incubated at room temperature in lysis buffer (50 mM Tris-HCl [pH = 7.9], 150 mM NaCl, 1 mM EDTA, 0.1% Nodinet P-40) for 15 min. The lysates were collected and stored briefly at −80°C.

Each lysate was mixed with 0.5 mL of sarcosyl 20% (Sigma) and 10 μL of 2-mercaptoethanol (Sigma), and PBS was added to a final volume of 10 mL. The mixture was transferred to a 14 mL polypropylene tube, and a cushion layer of PBS containing 30% sucrose and 0.01% BSA cushion was deposited at the bottom of the tube using a Pasteur pipette. After ultracentrifugation at 4°C in a Beckman SW40 rotor for 130 min at 40,000 rpm, the pellet was resuspended in 200 μL of PBS containing 0.01% BSA and centrifuged at 4°C for 5 min at 14,000 rpm. The supernatant was laid over 10.5 mL of a 7.5% to 45% linear sucrose gradient in PBS containing 0.01% BSA. This was followed by ultracentrifugation at 4°C in a SW40 rotor for 110 min at 40,000 rpm in a SW40 rotor. 0.4 mL fractions were collected from the top of the tube and kept at 4°C. 200 μL of each fraction were mixed with 1 mL of Optiphase HiSafe 3 (PerkinElmer), and the radioactivity was quantified using a liquid scintillation counter 1209 Rackbeta (Wallac).

Virion thermostability assays.

Virion suspensions with known titers were diluted in PBS containing 0.1% BSA to reach a final titer of 10³ PFU/mL in a volume of 2 mL. The diluted viral samples were incubated at 45°C in a calibrated thermoblock, and 0.6 mL aliquots were taken at 0, 45, and 120 min. Each aliquot was briefly kept at 4°C before being titrated as described previously. As an internal control, nonmutated WT virus was included in each experiment. The titers obtained for each time and mutant were averaged from duplicate titers obtained in each of two independent experiments.

Sequence and structural analyses and molecular graphics.

Sequence alignments were performed using the BLAST2 program (57). The programs WHATIF (58), VMD (59), UCSF Chimera (60), and the PDB atomic coordinates for the refined crystal structure of the HRV-B14 virion (4RHV) (26) were used to analyze the capsid structure, determine the structural parameters, interpret the results, and/or display the structural models.