A novel potassium channel encoded by Ectocarpus siliculosus virus

Kcv, the first identified viral potassium channel encoded by the green algae Paramecium bursaria chlorella virus (PBCV-1), conducted K(+) selective currents when expressed in heterologous systems. This K(+) channel was proposed to be important for PBCV-1 infection and replication. In the present study, we identified and functionally characterized a novel K(+) channel Kesv, encoded by Ectocarpus siliculosus virus that infects filamentous marine brown algae. Kesv encodes a protein of 124 amino acids and is 21.8% identical and 37.1% homologous to Kcv. Membrane topology programs predicted that Kesv consists of three transmembrane domains. When expressed in Xenopus oocytes, Kesv induced largely instantaneous, K(+) selective currents that were sensitive to block by Ba(2+) and amantadine. Thus, Kesv along with Kcv, constitutes an emerging family of viral potassium channels, which may play important roles in the life cycle of viruses.     

Viral adaptation to host: a proteome‐based analysis of codon usage and amino acid preferences

Viruses differ markedly in their specificity toward host organisms. Here, we test the level of general sequence adaptation that viruses display toward their hosts. We compiled a representative data set of viruses that infect hosts ranging from bacteria to humans. We consider their respective amino acid and codon usages and compare them among the viruses and their hosts. We show that bacteria‐infecting viruses are strongly adapted to their specific hosts, but that they differ from other unrelated bacterial hosts. Viruses that infect humans, but not those that infect other mammals or aves, show a strong resemblance to most mammalian and avian hosts, in terms of both amino acid and codon preferences. In groups of viruses that infect humans or other mammals, the highest observed level of adaptation of viral proteins to host codon usages is for those proteins that appear abundantly in the virion. In contrast, proteins that are known to participate in host‐specific recognition do not necessarily adapt to their respective hosts. The implication for the potential of viral infectivity is discussed.     

Potassium ion channels of Chlorella viruses cause rapid depolarization of host cells during infection

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.     

Discovery of Novel dsRNA Viral Sequences by In Silico Cloning and Implications for Viral Diversity, Host Range and Evolution

Genome sequence of viruses can contribute greatly to the study of viral evolution, diversity and the interaction between viruses and hosts. Traditional molecular cloning methods for obtaining RNA viral genomes are time-consuming and often difficult because many viruses occur in extremely low titers. DsRNA viruses in the families, Partitiviridae, Totiviridae, Endornaviridae, Chrysoviridae, and other related unclassified dsRNA viruses are generally associated with symptomless or persistent infections of their hosts. These characteristics indicate that samples or materials derived from eukaryotic organisms used to construct cDNA libraries and EST sequencing might carry these viruses, which were not easily detected by the researchers. Therefore, the EST databases may include numerous unknown viral sequences. In this study, we performed in silico cloning, a procedure for obtaining full or partial cDNA sequence of a gene by bioinformatics analysis, using known dsRNA viral sequences as queries to search against NCBI Expressed Sequence Tag (EST) database. From this analysis, we obtained 119 novel virus-like sequences related to members of the families, Endornaviridae, Chrysoviridae, Partitiviridae, and Totiviridae. Many of them were identified in cDNA libraries of eukaryotic lineages, which were not known to be hosts for these viruses. Furthermore, comprehensive phylogenetic analysis of these newly discovered virus-like sequences with known dsRNA viruses revealed that these dsRNA viruses may have co-evolved with respective host supergroups over a long evolutionary time while potential horizontal transmissions of viruses between different host supergroups also is possible. We also found that some of the plant partitiviruses may have originated from fungal viruses by horizontal transmissions. These findings extend our knowledge of the diversity and possible host range of dsRNA viruses and offer insight into the origin and evolution of relevant viruses with their hosts.     

Viral component of the human genome

Relationships between viruses and their human host are traditionally described from the point of view taking into consideration hosts as victims of viral aggression, which results in infectious diseases. However, these relations are in fact two-sided and involve modifications of both the virus and host genomes. Mutations that accumulate in the populations of viruses and hosts may provide them advantages such as the ability to overcome defense barriers of host cells or to create more efficient barriers to deal with the attack of the viral agent. One of the most common ways of reinforcing anti-viral barriers is the horizontal transfer of viral genes into the host genome. Within the host genome, these genes may be modified and extensively expressed to compete with viral copies and inhibit the synthesis of their products or modulate their functions in other ways. This review summarizes the available data on the horizontal gene transfer between viral and human genomes and discusses related problems.     

Vertically transmitted viral endosymbionts of insects: do sigma viruses walk alone?

Insects are host to a wide range of vertically transmitted bacterial endosymbionts, but we know relatively little about their viral counterparts. Here, we discuss the vertically transmitted viral endosymbionts of insects, firstly examining the diversity of this group, and then focusing on the well-studied sigma viruses that infect dipterans. Despite limited sampling, evidence suggests that vertically transmitted viruses may be common in insects. Unlike bacteria, viruses can be transmitted through sperm and eggs, a trait that allows them to rapidly spread through host populations even when infection is costly to the host. Work on Drosophila melanogaster has shown that sigma viruses and their hosts are engaged in a coevolutionary arms race, in which the spread of resistance genes in the host population is followed by the spread of viral genotypes that can overcome host resistance. In the long-term, associations between sigma viruses and their hosts are unstable, and the viruses persist by occasionally switching to new host species. It therefore seems likely that viral endosymbionts have major impacts on the evolution and ecology of insects.     

Evasion and exploitation of chemokines by viruses

Chemokines and chemokine receptors play a critical role in the host defense against viruses by mobilizing leukocytes to sites of infection, injury and inflammation. In order to replicate successfully within their host organisms, viruses have devised novel strategies for exploiting or subverting chemokine networks. This review summarizes various mechanisms that are currently known to be used by viruses for modulating chemokine activities including viral homologs of chemokines and chemokine receptors and soluble viral chemokine binding proteins. Insight into these strategies is providing a wealth of information on viral-host interactions, the function of chemokines in host defense and may help to generate novel anti-chemokine agents for treating against viral diseases or inflammatory disorders.     

Consequences of host adaptation for performance of vesicular stomatitis virus in novel thermal environments

Although it is widely assumed that the selective advantage of niche specialization drives species biodiversity, some theory suggests that generalists are favored over specialists when environments change unexpectedly. But this idea is rarely tested empirically, and its relevance is unknown for microparasites such as RNA viruses. Due to their small genome sizes pleiotropy is not uncommon in RNA viruses. Therefore, the genetic architectures underlying generalist traits may be indirectly molded by selection to better prepare generalist organisms for growth in new environments. Previously, vesicular stomatitis viruses were evolved to specialize on a single host, or to generalize on multiple hosts. Here we test whether virus generalists arising in the context of host adaptation also perform differently than specialists when viruses grow at novel temperatures. We compared thermal reaction norms of performance, within and among groups of viral specialists and generalists. Results showed that host adaptation was consequential for some fitness traits at novel temperatures due to modification of pleiotropic viral genes. Contrary to theoretical predictions, host generalists were selectively disadvantaged at extreme cool and warm environments. Multi-host adaptation may compromise the evolved thermostability of viral proteins, creating a cost of host generalization when viruses replicate at extreme temperatures.     

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.     

Biological and molecular events associated with simultaneous transmission of plant viruses by invertebrate and fungal vectors

Viruses are likely to be the most dangerous parasites of living organisms because of their widespread occurrence, possible deleterious effects on their hosts and high rates of evolution. Virus host‐to‐host transmission is a critical step in the virus life cycle, because it enables survival in a given environment and efficient dissemination. As hosts of plant viruses are not mobile, these pathogens have adopted diverse transmission strategies involving various vector organisms, mainly arthropods, nematodes, fungi and protists. In nature, plants are often infected with more than one virus at a time, thereby creating potential sources for vectors to acquire and transmit simultaneously two or more viruses. Simultaneous transmission can result in multiple infections of new host plants, which become subsequent potential sources of the viruses, thus enhancing the spread of the diseases caused by these pathogens. Moreover, it can contribute to the maintenance of viral genetic diversity in the host communities. However, despite its possible significance, the problem of the simultaneous transmission of plant viruses by vectors has not been investigated in detail. In this review, the current knowledge on multiple viral transmissions by aphids, whiteflies, leafhoppers, planthoppers, nematodes and fungi is outlined.     

Dimer Interface Migration in a Viral Sulfhydryl Oxidase

Large double-stranded DNA viruses, including poxviruses and mimiviruses, encode enzymes to catalyze the formation of disulfide bonds in viral proteins produced in the cell cytosol, an atypical location for oxidative protein folding. These viral disulfide catalysts belong to a family of sulfhydryl oxidases that are dimers of a small five-helix fold containing a Cys-X-X-Cys motif juxtaposed to a flavin adenine dinucleotide cofactor. We report that the sulfhydryl oxidase pB119L from African swine fever virus (ASFV) uses for self-assembly surface different from that observed in homologs from mammals, plants, and fungi. Within a protein family, different packing interfaces for the same oligomerization state are extremely rare. We find that the alternate dimerization mode seen in ASFV pB119L is not characteristic of all viral sulfhydryl oxidases, as the flavin-binding domain from a mimivirus sulfhydryl oxidase assumes the same dimer structure as the known eukaryotic enzymes. ASFV pB119L demonstrates the potential of large double-stranded DNA viruses, which have faster mutation rates than their hosts and the tendency to incorporate host genes, to pioneer new protein folds and self-assembly modes.     

Identification of putative potassium channel homologues in pathogenic protozoa

K(+) channels play a vital homeostatic role in cells and abnormal activity of these channels can dramatically alter cell function and survival, suggesting that they might be attractive drug targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, leishmaniasis, trypanosomiasis and dysentery that are responsible for millions of deaths each year worldwide. The genomes of many protozoan parasites have recently been sequenced, allowing rational design of targeted therapies. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of K(+) channels. These protozoan K(+) channel homologues represent novel targets for anti-parasitic drugs. Differences in the sequences and diversity of human and parasite proteins may allow pathogen-specific targeting of these K(+) channel homologues.     

Functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses

Chlorella viruses are a source of interesting membrane transport proteins. Here we examine a putative K(+) transporter encoded by virus FR483 and related chlorella viruses. The protein shares sequence and structural features with HAK/KUP/KT-like K(+) transporters from plants, bacteria and fungi. Yeast complementation assays and Rb(+) uptake experiments show that the viral protein, termed HAKCV (high-affinity K(+) transporter of chlorella virus), is functional, with transport characteristics that are similar to those of known K(+) transporters. Expression studies revealed that the protein is expressed as an early gene during viral replication, and proteomics data indicate that it is not packaged in the virion. The function of HAKCV is unclear, but the data refute the hypothesis that the transporter acts as a substitute for viral-encoded K(+) channels during virus infection.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.     

Generation of infectious molecular clones of simian immunodeficiency virus from fecal consensus sequences of wild chimpanzees

Studies of simian immunodeficiency viruses (SIVs) in their endangered primate hosts are of obvious medical and public health importance, but technically challenging. Although SIV-specific antibodies and nucleic acids have been detected in primate fecal samples, recovery of replication-competent virus from such samples has not been achieved. Here, we report the construction of infectious molecular clones of SIVcpz from fecal viral consensus sequences. Subgenomic fragments comprising a complete provirus were amplified from fecal RNA of three wild-living chimpanzees and sequenced directly. One set of amplicons was concatenated using overlap extension PCR. The resulting clone (TAN1.24) contained intact genes and regulatory regions but was replication defective. It also differed from the fecal consensus sequence by 76 nucleotides. Stepwise elimination of all missense mutations generated several constructs with restored replication potential. The clone that yielded the most infectious virus (TAN1.910) was identical to the consensus sequence in both protein and long terminal repeat sequences. Two additional SIVcpz clones were constructed by direct synthesis of fecal consensus sequences. One of these (TAN3.1) yielded fully infectious virus, while the second one (TAN2.69) required modification at one ambiguous site in the viral pol gene for biological activity. All three reconstructed proviruses produced infectious virions that replicated in human and chimpanzee CD4(+) T cells, were CCR5 tropic, and resembled primary human immunodeficiency virus type 1 isolates in their neutralization phenotype. These results provide the first direct evidence that naturally occurring SIVcpz strains already have many of the biological properties required for persistent infection of humans, including CD4 and CCR5 dependence and neutralization resistance. Moreover, they outline a new strategy for obtaining medically important "SIV isolates" that have thus far eluded investigation. Such isolates are needed to identify viral determinants that contribute to cross-species transmission and host adaptation.     

Relationships between polydnavirus genomes and viral gene expression

Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication.     

Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.     

病毒密码子使用频率研究进展

密码子使用频率的研究多集中在自养生物中,对于寄生生物如病毒的研究相对较少,近些年的研究表明某些病毒基因的密码子使用频率和宿主细胞不完全匹配,密码子使用在病毒和宿主的相互作用中起了重要的作用。本对病毒密码子使用频率的特点,影响病毒密码子使用频率的因素,几种典型病毒的密码子使用特点和密码子使用频率研究的方法做了论述。