Viral genome organization.
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Cryoelectron Microscopy↗
Biomedical subjects
B V Prasad
Publications and source records attributed to B V Prasad.
Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion.
The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.
Amino Acid Sequence↗
Structural studies on gastroenteritis viruses.
There are many recent advances in our understanding of the structure-function relationships in rotavirus, a major pathogen of infantile gastroenteritis, and Norwalk virus, a causative agent of epidemic gastroenteritis in humans. Rotavirus is a large (1000 A) and complex icosahedral assembly formed by three concentric capsid layers that enclose the viral genome of 11 dsRNA segments. Because of its medical relevance, intriguing structural complexity, and several unique strategies in the morphogenesis and replication, this virus has been the subject of extensive biochemical, genetic and structural studies. Using a combination of electron cryomicroscopy and computer image processing together with atomic resolution X-ray structural information, we have been able to provide not only a better description of the rotavirus architecture, but also a better understanding of the structural basis of various biological functions such as trypsin-enhanced infectivity, virus assembly and the dynamic process of endogenous transcription. In contrast to rotavirus, Norwalk virus has a simple architecture with an icosahedral capsid made of 180 copies of a single protein. We have determined the structure of the Norwalk virus capsid to a resolution of 3.4 A using X-ray crystallographic techniques. These studies have provided valuable information on domain organization in the capsid protein, and residues that may be critical for dimerization, assembly, strain-specificity and antigenicity.
Capsid↗
Genetic evidence on the origins of Indian caste populations.
The origins and affinities of the approximately 1 billion people living on the subcontinent of India have long been contested. This is owing, in part, to the many different waves of immigrants that have influenced the genetic structure of India. In the most recent of these waves, Indo-European-speaking people from West Eurasia entered India from the Northwest and diffused throughout the subcontinent. They purportedly admixed with or displaced indigenous Dravidic-speaking populations. Subsequently they may have established the Hindu caste system and placed themselves primarily in castes of higher rank. To explore the impact of West Eurasians on contemporary Indian caste populations, we compared mtDNA (400 bp of hypervariable region 1 and 14 restriction site polymorphisms) and Y-chromosome (20 biallelic polymorphisms and 5 short tandem repeats) variation in approximately 265 males from eight castes of different rank to approximately 750 Africans, Asians, Europeans, and other Indians. For maternally inherited mtDNA, each caste is most similar to Asians. However, 20%-30% of Indian mtDNA haplotypes belong to West Eurasian haplogroups, and the frequency of these haplotypes is proportional to caste rank, the highest frequency of West Eurasian haplotypes being found in the upper castes. In contrast, for paternally inherited Y-chromosome variation each caste is more similar to Europeans than to Asians. Moreover, the affinity to Europeans is proportionate to caste rank, the upper castes being most similar to Europeans, particularly East Europeans. These findings are consistent with greater West Eurasian male admixture with castes of higher rank. Nevertheless, the mitochondrial genome and the Y chromosome each represents only a single haploid locus and is more susceptible to large stochastic variation, bottlenecks, and selective sweeps. Thus, to increase the power of our analysis, we assayed 40 independent, biparentally inherited autosomal loci (1 LINE-1 and 39 Alu elements) in all of the caste and continental populations (approximately 600 individuals). Analysis of these data demonstrated that the upper castes have a higher affinity to Europeans than to Asians, and the upper castes are significantly more similar to Europeans than are the lower castes. Collectively, all five datasets show a trend toward upper castes being more similar to Europeans, whereas lower castes are more similar to Asians. We conclude that Indian castes are most likely to be of proto-Asian origin with West Eurasian admixture resulting in rank-related and sex-specific differences in the genetic affinities of castes to Asians and Europeans.
Adult↗
Trypsin cleavage stabilizes the rotavirus VP4 spike.
Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.
Amino Acid Sequence↗
A monoclonal antibody specific for reovirus outer-capsid protein sigma3 inhibits sigma1-mediated hemagglutination by steric hindrance.
Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.
Animals↗
Identification and characterization of a transcription pause site in rotavirus.
In rotavirus, transcription of the 11 double-stranded RNA genome segments occurs within the structurally intact subviral particle, and nascent transcripts are released through channels penetrating the two capsid layers at the icosahedral vertices. To gain insight into the early molecular events in transcription, we used high-resolution polyacrylamide gel electrophoresis to investigate the length distribution of transcription products at various times following initiation. We observed that, in the subviral particle under normal conditions, transcript initiation and capping are followed by a momentary pause in elongation after the addition of 6 to 7 nucleotides. In the absence of the capping reaction cofactor S-adenosylmethionine, conditions under which the rate of nucleotide incorporation is reduced, we observe a significant decrease in the ratio of paused to full-length transcripts. We propose that this pause site may represent the point at which specific molecular events take place to facilitate processive elongation. Furthermore, our results indicate that the presence of specific ligands on the viral surface, such as VP7 in the mature virion, inhibits polymerase function. From the perspective of the viral replication cycle, this inhibition may serve to ensure that transcription occurs with greatest efficiency only after the virus has entered the cytoplasm and assumed the form of a double-layered particle.
Electrophoresis, Polyacrylamide Gel↗
Mitochondrial DNA variation in Nicobarese Islanders.
The aboriginal populations living in the Nicobar Islands are hypothesized to be descendants of people who were part of early human dispersals into Southeast Asia. However, analyses of ethnographic histories, languages, morphometric data, and protein polymorphisms have not yet resolved which worldwide populations are most closely related to the Nicobarese. Thus, to explore the origins and affinities of the Nicobar Islanders, we analyzed mitochondrial DNA (mtDNA) hypervariable region 1 sequence data from 33 Nicobarese Islanders and compared their mtDNA haplotypes to those of neighboring East Asians, mainland and island Southeast Asians, Indians, Australian aborigines, Pacific Islanders, and Africans. Unique Nicobarese mtDNA haplotypes, including five Nicobarese mtDNA haplotypes linked to the COII/tRNA(Lys) 9-bp deletion, are most closely related to mtDNA haplotypes from mainland Southeast Asian Mon-Kmer-speaking populations (e.g., Cambodians). Thus, the dispersal of southern Chinese into mainland Southeast Asia may have included a westward expansion and colonization of the islands of the Andaman Sea.
Adult↗
Mechanism of genome transcription in segmented dsRNA viruses.
Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for organization in the viral core; (4) the role of the innermost capsid protein as a scaffold on which the core components of the transcription apparatus are assembled; and (5) the release of nascent mRNA transcripts through channels at the icosahedral vertices. The process of genome transcription in dsRNA viruses will become better understood as structural studies progress to higher resolution and as more viruses become amenable to study using site-directed mutagenesis coupled with viral reconstitution to generate recombinant particles having precise functional and structural changes. Future studies will dissect important intermolecular interactions required for efficient mRNA synthesis and will shed further light on the reasons for which the viral core must be structurally intact in order for transcription to occur efficiently. Structural studies of the capping enzymes at atomic resolution will reveal how multiple enzyme activities reside within a single polypeptide and how they act in concert to synthesize the 5' cap on the end of each mature transcript. Perhaps most interestingly, high resolution structural studies of actively transcribing virions will provide insight into the conformational changes that occur within the core during mRNA synthesis. Together, these studies will clarify the function of this complex macromolecular machine and will also shed additional light on the basic principles of virus architecture and assembly, as well as provide avenues for the design of antiviral therapies.
Animals↗
X-ray crystallographic structure of the Norwalk virus capsid.
Norwalk virus, a noncultivatable human calicivirus, is the major cause of epidemic gastroenteritis in humans. The first x-ray structure of a calicivirus capsid, which consists of 180 copies of a single protein, has been determined by phase extension from a low-resolution electron microscopy structure. The capsid protein has a protruding (P) domain connected by a flexible hinge to a shell (S) domain that has a classical eight-stranded beta-sandwich motif. The structure of the P domain is unlike that of any other viral protein with a subdomain exhibiting a fold similar to that of the second domain in the eukaryotic translation elongation factor-Tu. This subdomain, located at the exterior of the capsid, has the largest sequence variation among Norwalk-like human caliciviruses and is likely to contain the determinants of strain specificity and cell binding.
Amino Acid Sequence↗
Comparative structural analysis of transcriptionally competent and incompetent rotavirus-antibody complexes.
During genome transcription in rotavirus, as with many segmented double-stranded RNA viruses, mRNA is transcribed within the intact subviral particle and translocated through specific channels in the capsid. To understand how the conformation of the capsid affects the efficiency of transcriptional events in the viral core, we carried out a series of comparative structural and biochemical studies to characterize four different structural forms of the virus exhibiting differing transcriptional behavior. Two of these were virus-antibody complexes having contrasting transcriptional capabilities, and two were variant structural forms of the virus that exist during the life cycle and also exhibit contrasting transcriptional behavior. Three-dimensional structural studies using electron cryomicroscopy showed that the binding of one Fab (8H2/G5) does not affect the conformation of the capsid, and the efficiency of mRNA production is similar to that of the native subviral particle. The other Fab (2A11/E9) introduces conformational changes in the capsid similar to those seen in the transcriptionally incompetent mature particle. In both of the transcriptionally incompetent particle types, mRNA synthesis was arrested after limited elongation with the resulting oligonucleotide transcripts remaining trapped inside the particles. Our results indicate that the continuous translocation of nascent mRNA through the capsid is critical for efficient transcript elongation and that the blockage of translocation causes premature termination of transcription.
Antigen-Antibody Complex↗
Multiple origins of the mtDNA 9-bp deletion in populations of South India.
The origins and genetic affinities of the more than 500 tribal populations living in South Asia are widely disputed. This may reflect differential contributions that continental populations have made to tribal groups in South Asia. We assayed for the presence of the intergenic COII/tRNALys 9-bp deletion in human mtDNA in 646 individuals from 12 caste and 14 tribal populations of South India and compared them to individuals from Africa, Europe, and Asia. The 9-bp deletion is observed in four South Indian tribal populations, the Irula, Yanadi, Siddi, and Maria Gond, and in the Nicobarese. Length polymorphisms of the 9-bp motif are present in the Santal, Khonda Dora, and Jalari, all of whom live in a circumscribed region on the eastern Indian coast. Phylogenetic analyses of mtDNA control region sequence from individuals with the 9-bp deletion indicate that it has arisen independently in some Indian tribal populations. Other 9-bp deletion haplotypes are likely to be of Asian and African origin, implying multiple origins of the 9-bp deletion in South India. These results demonstrate varying genetic affinities of different South Indian tribes to continental populations and underscore the complex histories of the tribal populations living in South Asia.
Africa↗
The structure of a cypovirus and the functional organization of dsRNA viruses.
Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.
Bluetongue virus↗
Female gene flow stratifies Hindu castes.
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DNA, Mitochondrial↗
Structural localization of the E3 glycoprotein in attenuated Sindbis virus mutants.
We have determined the three-dimensional structures of the wild-type Sindbis virus and two of its mutants that retain the E3 sequence within PE2. Using difference imaging between these mutants and the wild-type virus, we have assigned a location for the 64-amino-acid sequence corresponding to E3 in the mutant spike complex. In the wild-type virus, the spike is composed of an E1-E2 heterotrimer. The E3 protein was found to protrude midway between the center of the spike complex and the tips. Based on these results and the work of others, we propose a distribution for the functional domains of the spike proteins within the structure of wild-type Sindbis virus. Within the structure of the virus, the E1 domains form the central portion of the spike complex, while the tips are formed by the E2 domains that flare out from the center of the complex. The structural similarity between these Sindbis virus mutants and Ross River virus suggests that E3 may also be present in the latter, which is also a member of the Alphavirus genus.
Animals↗
An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.
BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.
Bluetongue virus↗
Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles.
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Animals↗
Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.
Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.
Animals↗