Intercellular Transmission of Viral Populations with Vesicles

A common paradigm holds that during cell-to-cell transmission, viruses behave as lone soldiers. Recently, we discovered not only that enteroviruses are transmitted via vesicles as populations of viral particles but also that this type of transmission enhances their infection efficiency (Y. H. Chen et al., Cell 160:619–630, 2015). This mechanism could be advantageous for the overall fitness of the viral population, promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here, we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is widespread among different virus families and includes populations of both viral particles and naked viral genomes.     

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development.     

A hybrid stochastic-deterministic approach to explore multiple infection and evolution in HIV

To study viral evolutionary processes within patients, mathematical models have been instrumental. Yet, the need for stochastic simulations of minority mutant dynamics can pose computational challenges, especially in heterogeneous systems where very large and very small sub-populations coexist. Here, we describe a hybrid stochastic-deterministic algorithm to simulate mutant evolution in large viral populations, such as acute HIV-1 infection, and further include the multiple infection of cells. We demonstrate that the hybrid method can approximate the fully stochastic dynamics with sufficient accuracy at a fraction of the computational time, and quantify evolutionary end points that cannot be expressed by deterministic models, such as the mutant distribution or the probability of mutant existence at a given infected cell population size. We apply this method to study the role of multiple infection and intracellular interactions among different virus strains (such as complementation and interference) for mutant evolution. Multiple infection is predicted to increase the number of mutants at a given infected cell population size, due to a larger number of infection events. We further find that viral complementation can significantly enhance the spread of disadvantageous mutants, but only in select circumstances: it requires the occurrence of direct cell-to-cell transmission through virological synapses, as well as a substantial fitness disadvantage of the mutant, most likely corresponding to defective virus particles. This, however, likely has strong biological consequences because defective viruses can carry genetic diversity that can be incorporated into functional virus genomes via recombination. Through this mechanism, synaptic transmission in HIV might promote virus evolvability.     

High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.     

Virus, phage, transposon and their regulatory small non-coding RNAs

Many reports have been accumulated describing not a few microRNAs (miRNAs) in eukaryotes target viral genomes, whereas a number of viruses also encode miRNA genes. These small RNAs play important roles on viral infection and their replication. In germ cells, another small RNA, piRNA is reported to repress endogenous transposons. Furthermore, CRISPR RNA target virus/phage genomes in both archaea and bacteria. Therefore, small RNA is deeply involved in a broad range of biological defense systems. This system may be applied not only to control replication of viruses or phages but also provide implication on regulating the growth of microorganisms including pathogenic bacteria.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

Effect of Synaptic Transmission on Viral Fitness in HIV Infection

HIV can spread through its target cell population either via cell-free transmission, or by cell-to-cell transmission, presumably through virological synapses. Synaptic transmission entails the transfer of tens to hundreds of viruses per synapse, a fraction of which successfully integrate into the target cell genome. It is currently not understood how synaptic transmission affects viral fitness. Using a mathematical model, we investigate how different synaptic transmission strategies, defined by the number of viruses passed per synapse, influence the basic reproductive ratio of the virus, R₀, and virus load. In the most basic scenario, the model suggests that R₀ is maximized if a single virus particle is transferred per synapse. R₀ decreases and the infection eventually cannot be maintained for larger numbers of transferred viruses, because multiple infection of the same cell wastes viruses that could otherwise enter uninfected cells. To explain the relatively large number of HIV copies transferred per synapse, we consider additional biological assumptions under which an intermediate number of viruses transferred per synapse could maximize R₀. These include an increased burst size in multiply infected cells, the saturation of anti-viral factors upon infection of cells, and rate limiting steps during the process of synapse formation.     

Membrane-associated virus replication complexes locate to plant conducting tubes

It is generally accepted that in order to establish a systemic infection in a plant, viruses move from the initially infected cell to the vascular tissues by cell-to-cell movement through plasmodesmata (PD), and load into the vascular conducting tubes (i.e. phloem sieve elements and xylem vessel elements) for long-distance movement. The viral unit in these movements can be a virion or a yet-to-be-defined ribonucleic protein (RNP) complex. Using live-cell imaging, our laboratory has previously demonstrated that membrane-bound replication complexes move cell-to-cell during turnip mosaic virus (TuMV) infection. Our recent study shows that these membrane-bound replication complexes end up in the vascular conducting tubes, which is likely the case for potato virus X (PVX) also. The presence of TuMV-induced membrane complexes in xylem vessels suggests that viral components could also be found in other apoplastic regions of the plant, such as the intercellular space. This possibility may have implications regarding how we approach the study of plant innate immune responses against viruses.     

Multiple infection dynamics has pronounced effects on the fitness of RNA viruses

Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two‐fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka‐Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed.     

Viral enzymes

Viral genomes show unequalled diversity, ranging from single-stranded DNA to double-stranded RNA. Moreover, viruses can quickly adapt to the host's immune response and drug treatment. Although they tend to make optimal use of the host cell's reservoir of proteins, viruses need to carry some enzymatic functions with them, as they may not be available or accessible in the infected cell. Recently, progress has been made in our structural understanding of viral enzymes involved in all stages of the viral life cycle, which includes entry, hijack, replication and exit stages.     

Quasispecies spatial models for RNA viruses with different replication modes and infection strategies

Empirical observations and theoretical studies suggest that viruses may use different replication strategies to amplify their genomes, which impact the dynamics of mutation accumulation in viral populations and therefore, their fitness and virulence. Similarly, during natural infections, viruses replicate and infect cells that are rarely in suspension but spatially organized. Surprisingly, most quasispecies models of virus replication have ignored these two phenomena. In order to study these two viral characteristics, we have developed stochastic cellular automata models that simulate two different modes of replication (geometric vs stamping machine) for quasispecies replicating and spreading on a two-dimensional space. Furthermore, we explored these two replication models considering epistatic fitness landscapes (antagonistic vs synergistic) and different scenarios for cell-to-cell spread, one with free superinfection and another with superinfection inhibition. We found that the master sequences for populations replicating geometrically and with antagonistic fitness effects vanished at low critical mutation rates. By contrast, the highest critical mutation rate was observed for populations replicating geometrically but with a synergistic fitness landscape. Our simulations also showed that for stamping machine replication and antagonistic epistasis, a combination that appears to be common among plant viruses, populations further increased their robustness by inhibiting superinfection. We have also shown that the mode of replication strongly influenced the linkage between viral loci, which rapidly reached linkage equilibrium at increasing mutations for geometric replication. We also found that the strategy that minimized the time required to spread over the whole space was the stamping machine with antagonistic epistasis among mutations. Finally, our simulations revealed that the multiplicity of infection fluctuated but generically increased along time.     

Comprehensive analysis of endogenous bornavirus-like elements in eukaryote genomes

Bornaviruses are the only animal RNA viruses that establish a persistent infection in their host cell nucleus. Studies of bornaviruses have provided unique information about viral replication strategies and virus–host interactions. Although bornaviruses do not integrate into the host genome during their replication cycle, we and others have recently reported that there are DNA sequences derived from the mRNAs of ancient bornaviruses in the genomes of vertebrates, including humans, and these have been designated endogenous borna-like (EBL) elements. Therefore, bornaviruses have been interacting with their hosts as driving forces in the evolution of host genomes in a previously unexpected way. Studies of EBL elements have provided new models for virology, evolutionary biology and general cell biology. In this review, we summarize the data on EBL elements including what we have newly identified in eukaryotes genomes, and discuss the biological significance of EBL elements, with a focus on EBL nucleoprotein elements in mammalian genomes. Surprisingly, EBL elements were detected in the genomes of invertebrates, suggesting that the host range of bornaviruses may be much wider than previously thought. We also review our new data on non-retroviral integration of Borna disease virus.     

Control the Host Cell Cycle: Viral Regulation of the Anaphase-Promoting Complex

Viruses commonly manipulate cell cycle progression to create cellular conditions that are most beneficial to their replication. To accomplish this feat, viruses often target critical cell cycle regulators in order to have maximal effect with minimal input. One such master regulator is the large, multisubunit E3 ubiquitin ligase anaphase-promoting complex (APC) that targets effector proteins for ubiquitination and proteasome degradation. The APC is essential for cells to progress through anaphase, exit from mitosis, and prevent a premature entry into S phase. These far-reaching effects of the APC on the cell cycle are through its ability to target a number of substrates, including securin, cyclin A, cyclin B, thymidine kinase, geminin, and many others. Recent studies have identified several proteins from a number of viruses that can modulate APC activity by different mechanisms, highlighting the potential of the APC in driving viral replication or pathogenesis. Most notably, human cytomegalovirus (HCMV) protein pUL21a was recently identified to disable the APC via a novel mechanism by targeting APC subunits for degradation, both during virus infection and in isolation. Importantly, HCMV lacking both viral APC regulators is significantly attenuated, demonstrating the impact of the APC on a virus infection. Work in this field will likely lead to novel insights into viral replication and pathogenesis and APC function and identify novel antiviral and anticancer targets. Here we review viral mechanisms to regulate the APC, speculate on their roles during infection, and identify questions to be addressed in future studies.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

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.     

Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed DeltaAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of DeltaAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of DeltaAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of DeltaAd. IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small DeltaAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for DeltaAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. DeltaAd.IR vectors were produced at a yield of approximately 10(4) particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. DeltaAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.