Viral Protein Sequences Research Articles

Tobacco etch virus Infectivity in Capsicum Spp. Is Determined by a Maximum of Three Amino Acids in the Viral Virulence Determinant VPg

Potyvirus resistance in Capsicum spp. has been attributed to amino acid substitutions at the pvr1 locus that cause conformational shifts in eukaryotic translation initiation factor eIF4E. The viral genome-linked protein (VPg) sequence was isolated and compared from three Tobacco etch virus (TEV) strains, highly aphid-transmissible (HAT), Mex21, and N, which differentially infect Capsicum genotypes encoding Pvr1(+), pvr1, and pvr1(2). Viral chimeras were synthesized using the TEV-HAT genome, replacing HAT VPg with Mex21 or N VPg. TEV HAT did not infect pepper plants homozygous for either the pvr1 or pvr1(2) allele. However, the novel chimeric TEV strains, TEVHAT(Mex21-VPg) and TEV-HAT(N-VPg), infected pvr1 and pvr1(2) pepper plants, respectively, demonstrating that VPg is the virulence determinant in this pathosystem. Three dimensional structural models predicted interaction between VPg and the susceptible eIF4E genotype in every case, while resistant genotypes were never predicted to interact. To determine whether there is a correlation between physical interaction of VPg with eIF4E and infectivity, the effects of amino acid variation within VPg were assessed. Interaction between pvr1(2) eIF4E and N VPg was detected in planta, implying that the six amino acid differences in N VPg relative to HAT VPg are responsible for restoring the physical interaction and infectivity.

FluShuffle and FluResort: new algorithms to identify reassorted strains of the influenza virus by mass spectrometry

BackgroundInfluenza is one of the oldest and deadliest infectious diseases known to man. Reassorted strains of the virus pose the greatest risk to both human and animal health and have been associated with all pandemics of the past century, with the possible exception of the 1918 pandemic, resulting in tens of millions of deaths. We have developed and tested new computer algorithms, FluShuffle and FluResort, which enable reassorted viruses to be identified by the most rapid and direct means possible. These algorithms enable reassorted influenza, and other, viruses to be rapidly identified to allow prevention strategies and treatments to be more efficiently implemented.ResultsThe FluShuffle and FluResort algorithms were tested with both experimental and simulated mass spectra of whole virus digests. FluShuffle considers different combinations of viral protein identities that match the mass spectral data using a Gibbs sampling algorithm employing a mixed protein Markov chain Monte Carlo (MCMC) method. FluResort utilizes those identities to calculate the weighted distance of each across two or more different phylogenetic trees constructed through viral protein sequence alignments. Each weighted mean distance value is normalized by conversion to a Z-score to establish a reassorted strain.ConclusionsThe new FluShuffle and FluResort algorithms can correctly identify the origins of influenza viral proteins and the number of reassortment events required to produce the strains from the high resolution mass spectral data of whole virus proteolytic digestions. This has been demonstrated in the case of constructed vaccine strains as well as common human seasonal strains of the virus. The algorithms significantly improve the capability of the proteotyping approach to identify reassorted viruses that pose the greatest pandemic risk.

Characterization of Bean Necrotic Mosaic Virus: A Member of a Novel Evolutionary Lineage within the Genus Tospovirus

BackgroundTospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S (Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e.g., the determination of the viral protein sequences for enlightenment of their evolutionary history.Methodology/Principal FindingsBiological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus.Conclusion/SignificancesTaken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.

A time lag insensitive approach for estimating HIV-1 transmission direction

Identifying the direction of transmission in transmission pairs is important both for forensic investigations and for the monitoring of HIV epidemics, however, reliable methods are not yet available due to the long time lag between infection and sampling in most real cases. Based on bottleneck effect and coreceptor switching, we aimed at identifying an estimator from sequences of viral gp120 proteins to determine transmission direction between transmission pairs. The estimator should be changed with HIV transmission but was independent of disease progression in an individual. Here, we present a novel and reliable approach for identifying transmission direction. We derived a set of conserved patterns, called common patterns, from the sequences of viruses, which differed in their coreceptor usage. The number of unique common patterns in viral sequences decreased with transmission but remained almost constant with the progress of disease in an individual. We used this number as an estimator to determine transmission direction in 73 transmission pairs for which the transmission direction was already known. Our method predicted transmission direction with an accuracy of up to 94.5%. Of greater importance, our approach was not influenced by time lags between infection and sampling, and even transmission direction for transmission pairs with long time lags ranging from 2 years to more than 18 years were correctly determined. Our approach for accurately determining transmission direction between transmission pairs is irrespective of the time lag between infection and sampling, which means a promising applications prospect.

A Comparative Study of Human TLR 7/8 Stimulatory Trimer Compositions in Influenza A Viral Genomes

BackgroundVariation in the genomes of single-stranded RNA viruses affects their infectivity and pathogenicity in two ways. First, viral genome sequence variations lead to changes in viral protein sequences and activities. Second, viral genome sequence variation produces diversity at the level of nucleotide composition and diversity in the interactions between viral RNAs and host toll-like receptors (TLRs). A viral genome-typing method based on this type of diversity has not yet been established.Methodology/Principal FindingsIn this study, we propose a novel genomic trait called the “TLR stimulatory trimer composition” (TSTC) and two quantitative indicators, Score S and Score N, named “TLR stimulatory scores” (TSS). Using the complete genome sequences of 10,994 influenza A viruses (IAV) and 251 influenza B viruses, we show that TSTC analysis reveals the diversity of Score S and Score N among the IAVs isolated from various hosts. In addition, we show that low values of Score S are correlated with high pathogenicity and pandemic potential in IAVs. Finally, we use Score S and Score N to construct a logistic regression model to recognize IAV strains that are highly pathogenic or have high pandemic potential.Conclusions/Significance Results from the TSTC analysis indicate that there are large differences between human and avian IAV genomes (except for segment 3), as illustrated by Score S. Moreover, segments 1, 2, 3 and 4 may be major determinants of the stimulatory activity exerted on human TLRs 7 and 8. We also find that a low Score S value is associated with high pathogenicity and pandemic potential in IAV. The π value from the TSS-derived logistic regression model is useful for recognizing emerging IAVs that have high pathogenicity and pandemic potential.

Metagenomic Analysis of Viruses from Bat Fecal Samples Reveals Many Novel Viruses in Insectivorous Bats in China

Increasing data indicate that bats harbor diverse viruses, some of which cause severe human diseases. In this study, sequence-independent amplification and high-throughput sequencing (Solexa) were applied to the metagenomic analysis of viruses in bat fecal samples collected from 6 locations in China. A total of 8,746,417 reads with a length of 306,124,595 bp were obtained. Among these reads, 13,541 (0.15%) had similarity to phage sequences and 9,170 (0.1%) had similarity to eukaryotic virus sequences. A total of 129 assembled contigs (>100 nucleotides) were constructed and compared with GenBank: 32 contigs were related to phages, and 97 were related to eukaryotic viruses. The most frequent reads and contigs related to eukaryotic viruses were homologous to densoviruses, dicistroviruses, coronaviruses, parvoviruses, and tobamoviruses, a range that includes viruses from invertebrates, vertebrates, and plants. Most of the contigs had low identities to known viral genomic or protein sequences, suggesting that a large number of novel and genetically diverse insect viruses as well as putative mammalian viruses are transmitted by bats in China. This study provides the first preliminary understanding of the virome of some bat populations in China, which may guide the discovery and isolation of novel viruses in the future.

Camps 2.0: Exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/.

Assessment of Hepatitis C Virus Protein Sequences with Regard to Interferon/Ribavirin Combination Therapy Response in Patients with HCV Genotype 1b

Hepatitis C virus (HCV) infection is a major and rising global health problem, affecting about 170 million people worldwide. The current standard of care treatment with interferon alpha and ribavirin in patients with the genotype 1 infection, the most frequent genotype in the USA and Western Europe, leads to a successful outcome in only about 50% of individuals. Accurate prediction of hepatitis C treatment response is of great benefit to patients and clinicians. The informational spectrum method, a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied here for the identification of the conserved information of the HCV proteins that correlate with the combination therapy outcome. Among the HCV proteins that we have analyzed the informational property of the p7 of HCV genotype 1b was best related to the therapy outcome. On the basis of these results, a simple bioinformatics criterion that could be useful in assessment of the response of HCV-infected patients to the combination therapy has been proposed.

An outbreak of coxsackievirus A6 hand, foot, and mouth disease associated with onychomadesis in Taiwan, 2010

BackgroundIn 2010, an outbreak of coxsackievirus A6 (CA6) hand, foot and mouth disease (HFMD) occurred in Taiwan and some patients presented with onychomadesis and desquamation following HFMD. Therefore, we performed an epidemiological and molecular investigation to elucidate the characteristics of this outbreak.MethodsPatients who had HFMD with positive enterovirus isolation results were enrolled. We performed a telephone interview with enrolled patients or their caregivers to collect information concerning symptoms, treatments, the presence of desquamation, and the presence of nail abnormalities. The serotypes of the enterovirus isolates were determined using indirect immunofluorescence assays. The VP1 gene was sequenced and the phylogenetic tree for the current CA6 strains in 2010, 52 previous CA6 strains isolated in Taiwan from 1998 through 2009, along with 8 reference sequences from other countries was constructed using the neighbor-joining command in MEGA software.ResultsOf the 130 patients with laboratory-confirmed CA6 infection, some patients with CA6 infection also had eruptions around the perioral area (28, 22%), the trunk and/or the neck (39, 30%) and generalized skin eruptions (6, 5%) in addition to the typical presentation of skin eruptions on the hands, feet, and mouths. Sixty-six (51%) CA6 patients experienced desquamation of palms and soles after the infection episode and 48 (37%) CA6 patients developed onychomadesis, which only occurred in 7 (5%) of 145 cases with non-CA6 enterovirus infection (p < 0.001). The sequences of viral protein 1 of CA6 in 2010 differ from those found in Taiwan before 2010, but are similar to those found in patients in Finland in 2008.ConclusionsHFMD patients with CA6 infection experienced symptoms targeting a broader spectrum of skin sites and more profound tissue destruction, i.e., desquamation and nail abnormalities.

Original Findings Associated with Two Cases of Bovine Papular Stomatitis

Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

Enterovirus 68 among Children with Severe Acute Respiratory Infection, the Philippines

Enterovirus 68 (EV68) is a rare enterovirus associated with respiratory illness that, unlike other enteroviruses, has been identified only from respiratory specimens. We identified EV68 from respiratory specimens of children hospitalized with a diagnosis of severe pneumonia in Leyte, Republic of the Philippines. Twenty-one samples showed high similarity with EV68 by sequencing of 5' nontranslated region; 17 of these samples were confirmed as EV68 by sequencing of viral protein 1 capsid coding region. Most previously reported EV68 cases had been identified as sporadic cases. All 21 patients we identified had severe illness, and 2 died, possibly the first reported fatal cases associated with EV68 infection. Our study suggests that EV68 may be a possible causative agent of severe respiratory illnesses.

Porcine Rotavirus Closely Related to Novel Group of Human Rotaviruses

We determined nucleotide sequences and inferred amino acid sequences of viral protein (VP) 4, VP6, VP7, and nonstructural protein 4 genes of a porcine rotavirus strain (SKA-1) from Japan. The strain was closely related to a novel group of human rotavirus strains (B219 and J19).

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

A Similar Motif Shared by HIV-1 gp41 and TCRA Trans-Membrane Domains Exploited by the HIV Virus to Modulate T-Cell Proliferation

The transmembrane domains (TMDs) of the T-cell receptor (TCR) play an important role in the assembly of the receptor complex. Previous studies have shown that the fusion peptide (FP) of HIV-1 inhibits T-cell activation and by that suppress the immune response against the virus. FP gains this activity by specifically binding to the TMD of TCRα and interfering with the assembly of the TCR complex We utilized in-silico testing of a TMD sequence library derived from virus protein sequences, and pin-pointed a nine amino-acid motif shared by a group of different viruses; this motif resembles the transmembrane domain of the α-subunit of the T-cell receptor (TCRα). The highest similarity was found within SIV and HIV glycoprotein 41 TMD (gp41 TMD). Previous studies have shown that stable interactions between TCRα and CD3 are localized to this nine amino acid motif within TCRα, and a peptide derived from it interfered and intervened in the TCR function when added exogenously. By combining biophysical and biochemical approaches we found that the gp41 TMD peptide co-localizes with CD3 within the TCR complex and inhibits T-cell proliferation in vitro. We also found that the inhibitory mechanism of gp41 TMD differs from that of FP. Disassociated from HIV, the gp41 TMD molecule provides a novel mechanism for down regulating undesirable responses and might be be used as an immunotherapeutic tool.

HIV-1 gp41 and TCRα Trans-Membrane Domains Share a Motif Exploited by the HIV Virus to Modulate T-Cell Proliferation

Viruses have evolved several strategies to modify cellular processes and evade the immune response in order to successfully infect, replicate, and persist in the host. By utilizing in-silico testing of a transmembrane sequence library derived from virus protein sequences, we have pin-pointed a nine amino-acid motif shared by a group of different viruses; this motif resembles the transmembrane domain of the α-subunit of the T-cell receptor (TCRα). The most striking similarity was found within the immunodeficiency virus (SIV and HIV) glycoprotein 41 TMD (gp41 TMD). Previous studies have shown that stable interactions between TCRα and CD3 are localized to this nine amino acid motif within TCRα, and a peptide derived from it (TCRα TMD, GLRILLLKV) interfered and intervened in the TCR function when added exogenously. We now report that the gp41 TMD peptide co-localizes with CD3 within the TCR complex and inhibits T cell proliferation in vitro. However, the inhibitory mechanism of gp41 TMD differs from that of the TCRα TMD and also from the other two known immunosuppressive regions within gp41.

Discriminating antigen and non-antigen using proteome dissimilarity II: viral and fungal antigens

Immunogenicity arises via many synergistic mechanisms, yet the overall dissimilarity of pathogenic proteins versus the host proteome has been proposed as a key arbiter. We have previously explored this concept in relation to Bacterial antigens; here we extend our analysis to antigens of viral and fungal origin. Sets of known viral and fungal antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we could not determine a threshold able meaningfully to separate non-antigen from antigen. We conclude that viral and fungal antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to mammalian genomes.

Proteomics search of influenza A viruses for adaptive mutations to human hosts

Evaluation of: Miotto O, Heiny AT, Albrecht R et al. Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains. PLoS ONE 5(2), e9025 (2010).The emergence of an influenza pandemic is of great concern globally. It is, therefore, necessary to have a better understanding of the adaptation of influenza A viruses to humans. The mutation patterns affecting host tropism may provide information on the mechanisms and determinants of the host barrier. The work by Miotto et al. describes a catalog of mutations observed specifically in human influenza A viruses by analyzing almost 100,000 influenza A virus protein sequences. These sites may be important for host tropism and characteristic mutations of human influenza viruses may be required for efficient human-to-human transmission. The catalog can be used for genetic surveillance of zoonotic strains of the influenza A virus to determine their pandemic potential, as well as for basic research on the influenza A virus.

‘Let the phage do the work’: Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980; Newcomb et al., 1999). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine (Earnshaw, Hendrix, and King, 1980; Heymann et al., 2003). DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

The persistence of infectious pancreatic necrosis virus and its influence on the early immune response

Persistent infection by IPNV was induced in RTG-2 and RTG-P1 cells in vitro and the influence of this phenomenon on viral infectivity, viral antigen expression and interference with homologous and heterologous viruses was characterized over successive passages. The induction of IFN was also assessed, as was the sequence of the VP2 viral capsid protein, the region believed to be responsible for virulence, attenuation or persistence. Viral antigen expression was recorded in cells with no evidence of cytopathic effects and in these conditions, flow cytometry was more sensitive than RT-PCR to demonstrate the presence of a non-lytic virus. Interference of homologous viral infection could be detected in cross-infection experiments and in RTG-P1 cells persistently infected with IPNV, the Mx1 promoter could still be activated for at least 5 successive passages. Indeed, although over-induction of luciferase was not observed by re-infection with homologous or heterologous viruses, a significant increase in luciferase was induced by poly I:C. IFN transcripts could be quantified by qRT-PCR in the persistent cells at several passages, suggesting that IFN plays a role in maintaining IPNV persistence. In addition, we observed the same determinants in the VP2 sequences from the persistent virus as those described previously for IPNV adaptation and persistence in cell culture.

Analysis of cassava brown streak viruses reveals the presence of distinct virus species causing cassava brown streak disease in East Africa

Cassava brown streak virus (CBSV) isolates were analysed from symptomatic cassava collected between 1997 and 2008 in the major cultivation regions of East Africa. An analysis of complete RNA genomes of seven isolates from Kenya, Tanzania, Mozambique, Uganda and Malawi revealed a common genome structure, but the isolates clearly clustered in two distinct clades. The first comprised isolates from Kenya, Uganda, Malawi, north-western Tanzania and the CBSV described previously, and shared between 87 and 95% nucleotide sequence identity, whilst the second included isolates from coastal regions of Mozambique and Tanzania, which shared only 70% nucleotide sequence identities with isolates of the first clade. When the amino acid sequences of viral proteins were compared, identities as low as 47% (Ham1) and 59% (P1) between the two clades were found. An antiserum obtained against the capsid protein of a clade 1 isolate identified a 43 kDa protein in clade 1 isolates and a 45 kDa protein in clade 2 isolates. Several cassava cultivars were susceptible to isolates of clade 2 but resistant to those of clade 1. The differences observed both in biological behaviour and in genomic and protein sequences indicate that cassava brown streak disease in East Africa is caused by at least two distinct virus species. It is suggested that those of clade 1 retain the species name Cassava brown streak virus, whilst those of clade 2 be classified as Cassava brown streak Mozambique virus.