Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.     

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.     

The Multiplicity of Infection of a Plant Virus Varies during Colonization of Its Eukaryotic Host

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.Virus evolution has been a very active area of research in the last few decades, as viruses are both important pathogens of humans, animals, and plants and good models to experimentally test hypotheses on parasite evolution or, more generally, central questions on evolutionary biology (11, 12, 21, 36). Considerable efforts have been devoted to modeling the evolution of viral populations. However, contrasting the theoretical models with reality may be hindered by limited experimental information on important parameters of the virus life cycle. The multiplicity of infection (MOI), i.e., the number of virus particles or genomes that may infect a cell, is a key parameter in many models of virus evolution (5, 6, 14, 15, 37, 38, 39, 52, 53, 57, 61) for which experimental estimates are scant.When a cell is coinfected by different viral genomes, competition may lead to decreased fitness of individual genotypes in comparison with their fitness in single infections (15, 31, 40). Thus, limiting coinfection may result in a selective advantage for viruses (58), which have developed mechanisms to prevent superinfection of previously infected cells (51, 60). On the other hand, infection of a cell by more than one virus genome is a prerequisite for two central phenomena in virus genetics to take place: recombination and complementation of defective mutants. Recombination between viral strains during replication in the same cell and complementation of defective mutants have been extensively documented for viruses infecting prokaryotes, animals, and plants (2, 25, 56), indicating that there must be some degree of coinfection and, hence, that the MOI must be higher than one in at least some infected cells. However, estimates of MOI in the natural hosts of viruses are surprisingly scarce in spite of this parameter''s relevance: values of about 2 to 3 have been reported for different DNA or RNA bacteriophages (26, 41, 51, 58), and a value of 4 to 5 was reported for Autographa californica nuclear polyhedrosis virus infecting larvae of the moth Tricoplusia ni (3), to our knowledge, the only estimate for a virus in its eukaryotic host. We are not aware of estimates reported for viruses infecting mammals or plants, although a MOI of about 3 can be inferred from the number of proviral copies of HIV in spleen cells of infected patients (29). This paucity of data may be due to the technical difficulty of directly measuring MOI, particularly within a eukaryotic host. Genetic approaches may provide valid alternatives for estimating MOI levels (3, 58), and here, the MOI of a plant virus is estimated through the analysis of the relative frequencies of two genotypes during the process of host colonization.Host colonization by plant-infecting viruses has been known for a long time to be a two-step phenomenon. First, colonization proceeds slowly from the initially infected cells to their neighbors by way of the cytoplasmic connections called plasmodesmata, a process known as cell-to-cell movement. After infection thus reaches the cells in the vasculature, the second step, known as long-distance or systemic movement, occurs as viruses move faster to distant organs through the vascular tissue, the phloem in most cases (59). As a result of these processes, the virus population within the infected plant may be strongly structured. Analyses of different viruses in different host plant species have shown that systemic movement causes population bottlenecks that may be severe (16, 28, 32, 34, 46), resulting in differences in the genetic composition of the virus subpopulations in different systemically infected organs. No analysis of population bottlenecks during cell-to-cell movement has been reported, but data indicate that the virus population within a leaf has a strong spatial structure with a separate distribution of different genotypes in different leaf areas. These reports derive from analyses of viruses that differ in genomic organization and gene expression strategies in different host plant species (9, 10, 23, 55); they indicate that a separate distribution of viral genotypes within the infected leaf is a general phenomenon and suggest limitation of coinfection. Data on the spatial exclusion of virus genotypes within the infected leaf are in apparent contradiction with the abundant evidence of recombination and complementation of defective mutants, which has been widely documented for plant viruses (19, 44, 50, 62). It should be pointed out that all reports on the spatial exclusion of virus genotypes in an infected leaf derive from microscopy observations, mostly at late times after infection of the tissue. No information is available on the kinetics of leaf colonization by viruses, and current data do not allow the estimation of MOI.In this report, we estimate the MOI of a plant RNA virus, Tobacco mosaic virus (TMV), in its systemic host, Nicotiana benthamiana. For this, we have reexamined the process of virus colonization by monitoring the progress of infection of two TMV genotypes in inoculated and in systemically infected leaves. The two TMV genotypes differed in the expression of fluorescent tags, either the green fluorescent protein (GFP) from Aequorea victoria (42, 43) or a red fluorescent protein (RFP) from Discosoma sp. (49). The expression of GFP and RFP allowed the precise quantification of the number of cells infected by either one or both TMV genotypes, and these data allowed the estimation of genotype frequencies and of MOI. The results show evidence of strong spatial structure of the virus population, with most cells being infected by either TMV-GFP or TMV-RFP alone and only a small fraction of cells being double infected. The kinetics of the single and double infections show that the MOI changes with time, decreasing as colonization progresses and therefore suggesting that exclusion mechanisms operate at later times after infection.     

Global transcriptional responses of Pseudomonas aeruginosa to phage PRR1 infection

The infectious cycles of viruses are known to cause dramatic changes to host cell function. The development of microarray technology has provided means to monitor host cell responses to viral infection at the level of global changes in mRNA levels. We have applied this methodology to investigate gene expression changes caused by a small, icosahedral, single-stranded-RNA phage, PRR1 (a member of the Leviviridae family), on its host, Pseudomonas aeruginosa, at different times during its growth cycle. Viral infection in this system resulted in changes in expression levels of <4% of P. aeruginosa genes. Interestingly, the number of genes affected by viral infection was significantly lower than the number of genes affected by changes in growth conditions during the experiment. Compared with a similar study that focused on the complex, double-stranded-DNA bacterial virus PRD1, it was evident that there were no universal responses to viral infection. However, in both cases, translation was affected in infected cells.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

P58(IPK), a plant ortholog of double-stranded RNA-dependent protein kinase PKR inhibitor,functions in viral pathogenesis

P58(IPK) is a cellular inhibitor of the mammalian double-stranded RNA-activated protein kinase (PKR). Here we provide evidence for the existence of its homolog in plants and its role in viral infection at the organism level. Viral infection of P58(IPK)-silenced Nicotiana benthamiana and Arabidopsis knockouts leads to host death. This host cell death is associated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF-2alpha). Loss of P58(IPK) leads to reduced virus titer, suggesting that wild-type P58(IPK) protein plays an important role in viral pathogenesis. Although our complementation results using mammalian P58(IPK) suggest conservation of the P58(IPK) pathway in plants and animals, its biological significance seems to be different in these two systems. In animals, P58(IPK) is recruited by the influenza virus to limit PKR-mediated innate antiviral response. In plants, P58(IPK) is required by viruses for virulence and therefore functions as a susceptibility factor.     

Viral Bimolecular Fluorescence Complementation: A Novel Tool to Study Intracellular Vesicular Trafficking Pathways

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.     

Multiplicity of infection does not accelerate infectivity evolution of viral parasites in laboratory microcosms

Coinfection with multiple parasite genotypes [multiplicity of infection (MOI)] creates within-host competition and opportunities for parasite recombination and is therefore predicted to be important for both parasite and host evolution. We tested for a difference in the infectivity of viral parasites (lytic phage Φ2) and resistance of their bacterial hosts (Pseudomonas fluorescens SBW25) under both high and low MOI during coevolution in laboratory microcosms. Results show that MOI has no effect on infectivity and resistance evolution during coevolution over ～80 generations of host growth, and this is true when the experiment is initiated with wild-type viruses and hosts, or with viruses and hosts that have already been coevolving for ～330 generations. This suggests that MOI does not have a net effect of accelerating parasite adaptation to hosts through recombination, or slowing adaptation to hosts through between-parasite conflict in this system.     

Fitness analyses of vesicular stomatitis strains with rearranged genomes reveal replicative disadvantages

Gene expression of the nonsegmented negative-strand RNA viruses is determined by the position of each gene relative to that the single 3' promoter. The general order of genes among all of the viruses of the order Mononegavirales is highly conserved. In previous work we generated recombinant viruses in which the order of the three central genes of the prototypical rhabdovirus, vesicular stomatitis virus, was rearranged to all six possible permutations. While some of these viruses replicated less well than the wild type when assayed by single-step growth analyses in BSC-1 cells, others replicated as well or slightly better. In the work reported here, we used competition assays to compare the fitness of the viruses with alternative gene orders to that of the wild-type (wt) virus. We found that the relative fitness of these recombinant viruses depended on the multiplicity of infection (MOI) but not on the population size. However, during competitions at low MOI, when complementation cannot compensate for the defects of the populations with rearranged genomes, the virus with the wt gene order was always the most fit.     

Evolutionary dynamics of genome segmentation in multipartite viruses

Multipartite viruses are formed by a variable number of genomic fragments packed in independent viral capsids. This fact poses stringent conditions on their transmission mode, demanding, in particular, a high multiplicity of infection (MOI) for successful propagation. The actual advantages of the multipartite viral strategy are as yet unclear. The origin of multipartite viruses represents an evolutionary puzzle. While classical theories suggested that a faster replication rate or higher replication fidelity would favour shorter segments, recent experimental results seem to point to an increased stability of virions with incomplete genomes as a factor able to compensate for the disadvantage of mandatory complementation. Using as main parameters differential stability as a function of genome length and MOI, we calculate the conditions under which a set of complementary segments of a viral genome would outcompete the non-segmented variant. Further, we examine the likeliness that multipartite viral forms could be the evolutionary outcome of the competition among the defective genomes of different lengths that spontaneously arise under replication of a complete, wild-type genome. We conclude that only multipartite viruses with a small number of segments could be produced in our scenario, and discuss alternative hypotheses for the origin of multipartite viruses with more than four segments.     

Replication kinetics of coxsackievirus A16 in human rhabdomyosarcoma cells

Coxsackievirus A16 (CVA16), together with enterovirus type 71 (EV71), is responsible for most cases of hand, foot and mouth disease (HFMD) worldwide. Recent findings suggest that the recombination between CVA16 and EV71, and the co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years. It is therefore important to further understand the virology, epidemiology, virus-host interactions and host pathogenesis of CVA16. In this study, we describe the viral kinetics of CVA16 in human rhabdomyosarcoma (RD) cells by analyzing the cytopathic effect (CPE), viral RNA replication, viral protein expression, viral RNA package and viral particle secretion in RD cells. We show that CVA16 appears to first attach, uncoat and enter into the host cell after adsorption for 1 h. Later on, CVA16 undergoes rapid replication from 3 to 6 h at MOI 1 and until 9 h at MOI 0.1. At MOI 0.1, CVA16 initiates a secondary infection as the virions were secreted before 9 h p.i. CPE was observed after 12 h p.i., and viral antigen was first detected at 6 h p.i. at MOI 1 and at 9 h p.i. at MOI 0.1. Thus, our study provides important information for further investigation of CVA16 in order to better understand and ultimately control infections with this virus.     

The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.     

Quantitative subcellular proteome and secretome profiling of influenza A virus-infected human primary macrophages

Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X₇ receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages.     

Adeno-associated virus site-specific integration and AAVS1 disruption

Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.