The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.     

MicroRNA Antagonism of the Picornaviral Life Cycle: Alternative Mechanisms of Interference

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3′ untranslated region (3′UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.     

Evolution of mutational robustness in an RNA virus

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage φ6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.     

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.     

Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.     

A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.     

Modeling host-associating microbes under selection

The concept of fitness is often reduced to a single component, such as the replication rate in a given habitat. For species with multi-step life cycles, this can be an unjustified oversimplification, as every step of the life cycle can contribute to the overall reproductive success in a specific way. In particular, this applies to microbes that spend part of their life cycles associated to a host. In this case, there is a selection pressure not only on the replication rates, but also on the phenotypic traits associated to migrating from the external environment to the host and vice-versa (i.e., the migration rates). Here, we investigate a simple model of a microbial lineage living, replicating, migrating and competing in and between two compartments: a host and an environment. We perform a sensitivity analysis on the overall growth rate to determine the selection gradient experienced by the microbial lineage. We focus on the direction of selection at each point of the phenotypic space, defining an optimal way for the microbial lineage to increase its fitness. We show that microbes can adapt to the two-compartment life cycle through either changes in replication or migration rates, depending on the initial values of the traits, the initial distribution across the two compartments, the intensity of competition, and the time scales involved in the life cycle versus the time scale of adaptation (which determines the adequate probing time to measure fitness). Overall, our model provides a conceptual framework to study the selection on microbes experiencing a host-associated life cycle.Subject terms: Microbial ecology, Symbiosis, Theoretical ecology, Evolution, Microbiome     

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.     

Trade-offs in resource allocation in the intracellular life-cycle of hepatitis C virus

Positive sense single-stranded RNA viruses undergo three mutually exclusive processes to replicate within a cell. These are translation to produce proteins, replication to produce RNA viral genomes, and packaging to form virions. The allocation of newly synthesised viral genomes to these processes, which can be regarded as life-history traits, may be subject to natural selection for efficient reproduction. Here, we develop a mathematical model of the process of intracellular viral replication to study alternative strategies for the allocation and reallocation of viral genomes to these processes. We explore four cases of the model: (1) Free Movement, in which viral genomes can freely be allocated and reallocated among translation, replication and packaging; (2) Unidirectional Reallocation, in which allocation occurs freely but reallocation can only proceed from translation to replication to packaging; (3) Conveyor Belt, in which viral genomes are first allocated to translation, then passed on to replication and finally to packaging; and (4) Permanent Allocation in which new genomes are allocated to the three processes but not reallocated between them. We apply this model to hepatitis C virus and study changes in the production of virus as the rates of allocation and reallocation are varied. We find that high viral production occurs when allocation and reallocation of the genome are weighted towards the translation and replication processes. The replication process in particular is favoured. The most productive strategy is a form of the Free Movement model in which genomes are allocated entirely to the replication-translation cycle while allowing some genomes to be packaged through reallocation.     

Constraints to genetic exchange support gene coadaptation in a tripartite RNA virus

Genetic exchange by recombination, or reassortment of genomic segments, has been shown to be an important process in RNA virus evolution, resulting often in important phenotypic changes affecting host range and virulence. However, data from numerous systems indicate that reassortant or recombinant genotypes could be selected against in virus populations and suggest that there is coadaptation among viral genes. Little is known about the factors affecting the frequency of reassortants and recombinants along the virus life cycle. We have explored this issue by estimating the frequency of reassortant and recombinant genotypes in experimental populations of Cucumber mosaic virus derived from mixed infections with four different pairs of isolates that differed in about 12% of their nucleotide sequence. Genetic composition of progeny populations were analyzed at various steps of the virus life cycle during host colonization: infection of leaf cells, cell-to-cell movement within the inoculated leaf, encapsidation of progeny genomes, and systemic movement to upper noninoculated leaves. Results indicated that reassortant frequencies do not correspond to random expectations and that selection operates against reassortant genotypes. The intensity of selection, estimated through the use of log-linear models, increased as host colonization progressed. No recombinant was detected in any progeny. Hence, results showed the existence of constraints to genetic exchange linked to various steps of the virus life cycle, so that genotypes with heterologous gene combinations were less fit and disappeared from the population. These results contribute to explain the low frequency of recombinants and reassortants in natural populations of many viruses, in spite of high rates of genetic exchange. More generally, the present work supports the hypothesis of coadaptation of gene complexes within the viral genomes.     

Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.     

Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions.

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.     

Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

Background

Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.

Principal Findings

We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.

Conclusions

Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.     

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.