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Herpesvirus genomes enter the eukaryotic nucleus as large linear double stranded DNA molecules that are free of any proteins (naked DNA). Once inside the nucleus, the HSV-1 genomes immediately associate with proteins that will be instrumental in the organization and regulation of these genomes. These initial interactions are thought to determine the fate of the infecting genomes. In general, the host cell has evolved several mechanisms to suppress viral genomes and induce latent or abortive infections. On the other hand, the virus has evolved to use viral and cellular factors to promote lytic infection. Recent findings suggest that not all viral genomes in the infected nucleus will develop progeny and that not all genetically identical cells will support successful virus propagation. Thus, the decision between different fates of infection is determined at both single-cell and single-genome levels. Here we summarize current knowledge on the conditions and interactions that lead to each outcome and discuss the unknown determinants.

Homologous recombination (HR) is considered a major driving force of evolution because it generates and expands genetic diversity. Evidence of HR between coinfecting herpesvirus DNA genomes can be found frequently both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when coinfecting viral genomes interact. To study the spatiotemporal requirements for intergenomic recombination, we developed an assay with dual-color FISH that enables detection of HR between different pairs of coinfecting HSV-1 genomes. Our results revealed that HR increases intermingling of RCs derived from different genomes. Furthermore, inhibition of RC movement reduces the rate of HR events among coinfecting viruses. Finally, we observed correlation between nuclear size and the number of RCs per nucleus. Our findings suggest that both viral replication and recombination are subject to nuclear spatial constraints. Other DNA viruses and cellular DNA are likely to encounter similar restrictions.—Tomer, E., Cohen, E. M., Drayman, N., Afriat, A., Weitzman, M. D., Zaritsky, A., Kobiler, O. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19., Author summary The correlates of effective durable antibody response to SARS-CoV-2 infection are still unclear. In this study, we compared B cell receptor signatures in 8 Severe versus 10 Mild SARS-CoV-2 infected Israeli donors, at 1.5 months post infection using molecular and bioinformatic approaches. We found distinct features between the two groups with higher anti-SARS-CoV-2 receptor binding domain (RBD) plasma IgG titers and increased B cell expansion in donors with severe disease manifestations. We further isolated 22 monoclonal antibodies from these donors, 6 of which were highly potent neutralizing the live virus and inhibited the fusion of infected cells. Using mutagenesis and peptide libraries we mapped the binding sites of the neutralizing antibodies on the RBD of the SARS-CoV-2 Spike. We next demonstrated that combinations of different classes of neutralizing mAbs can completely block the live virus from spreading in culture. Lastly, we performed a bioinformatic search in 49 healthy BCR repertoires identifying precursors for these neutralizing antibodies in the top 30 most common precursors, suggesting that these antibodies can be readily produced by the majority of the uninfected population upon antigenic stimulation.

Homologous recombination (HR) is considered a major driving force of evolution since it generates and expands genetic diversity. Evidence of HR between co-infecting herpesvirus DNA genomes can be found frequently, bothin vitroand in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when co-infecting viral genomes interact. To study the spatiotemporal requirements for inter-genomic recombination, we developed an assay with dual-color fluorescencein situhybridization which enables detection of HR between different pairs of co-infecting HSV-1 genomes. Our results revealed that when viral RCs enlarge towards each other, there is detectable overlap between territories of genomes from each virus. Infection with paired viruses that allow visualization of HR correlates with increased overlap of RCs. Further, inhibition of RC movement reduces the rate of HR events among co-infecting viruses. Taken together, these findings suggest that inter-genomic HR events take place during replication of HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce. Our observations have implications on understanding the recombination restrictions of other DNA viruses and cellular DNA.

Synchronous viral infection facilitates the study of viral gene expression, viral host interactions, and viral replication processes. However, the protocols for achieving synchronous infections were hardly ever tested in proper temporal resolution at the single-cell level. We set up a fluorescence-based, time lapse microscopy assay to study sources of variability in the timing of gene expression during herpes simplex virus-1 (HSV-1) infection. We found that with the common protocol, the onset of gene expression within different cells can vary by more than 3 h. We showed that simultaneous viral genome entry to the nucleus can be achieved with a derivative of the previously characterized temperature sensitive mutant tsB7, however, this did not improve gene expression synchrony. We found that elevating the temperature in which the infection is done and increasing the multiplicity of infection (MOI) significantly promoted simultaneous onset of viral gene expression among infected cells. Further, elevated temperature result in a decrease in the coefficient of variation (a standardized measure of dispersion) of viral replication compartments (RCs) sizes among cells as well as a slight increment of viral late gene expression synchrony. We conclude that simultaneous viral gene expression can be improved by simple modifications to the infection process and may reduce the effect of single-cell variability on population-based assays.

The cellular response to viral infection is usually studied at the level of cell populations. Currently, it remains an open question whether and to what extent cell-to-cell variability impacts the course of infection. Here we address this by dynamic proteomics—imaging and tracking 400 yellow fluorescent protein (YFP)-tagged host proteins in individual cells infected by herpes simplex virus 1. By quantifying time-lapse fluorescence imaging, we analyze how cell-to-cell variability impacts gene expression from the viral genome. We identify two proteins, RFX7 and geminin, whose levels at the time of infection correlate with successful initiation of gene expression. These proteins are cell cycle markers, and we find that the position in the cell cycle at the time of infection (along with the cell motility and local cell density) can reasonably predict in which individual cells gene expression from the viral genome will commence. We find that the onset of cell division dramatically impacts the progress of infection, with 70% of dividing cells showing no additional gene expression after mitosis. Last, we identify four host proteins that are specifically modulated in infected cells, of which only one has been previously recognized. SUMO2 and RPAP3 levels are rapidly reduced, while SLTM and YTHDC1 are redistributed to form nuclear foci. These modulations are dependent on the expression of ICP0, as shown by infection with two mutant viruses that lack ICP0. Taken together, our results provide experimental validation for the long-held notion that the success of infection is dependent on the state of the host cell at the time of infection. IMPORTANCE High-throughput assays have revolutionized many fields in biology, both by allowing a more global understanding of biological processes and by deciphering rare events in subpopulations. Here we use such an assay, dynamic proteomics, to study viral infection at the single-cell level. We follow tens of thousands of individual cells infected by herpes simplex virus using fluorescence live imaging. Our results link the state of a cell at the time of virus infection with its probability to successfully initiate gene expression from the viral genome. Further, we identified three cellular proteins that were previously unknown to respond to viral infection. We conclude that dynamic proteomics provides a powerful tool to study single-cell differences during viral infection.

Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the infection. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may down regulate the number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.

Respiratory syncytial virus (RSV) is the most common cause of US infant hospitalization. Additionally, RSV is responsible for 10,000 deaths annually among the elderly across the United States, and accounts for nearly as many hospitalizations as influenza. Currently, several RSV vaccine candidates are under development to target different age groups. To evaluate the potential effectiveness of age-specific vaccination strategies in averting RSV incidence, we developed a transmission model that integrates data on daily infectious viral load and changes of behavior associated with RSV symptoms. Calibrating to RSV weekly incidence rates in Texas, California, Colorado, and Pennsylvania, we show that in all states considered, an infected child under 5 y of age is more than twice as likely as a person over 50 y of age to transmit the virus. Geographic variability in the effectiveness of a vaccination program across states arises from interplay between seasonality patterns, population demography, vaccination uptake, and vaccine mechanism of action. Regardless of these variabilities, our analysis showed that allocating vaccine to children under 5 y of age would be the most efficient strategy per dose to avert RSV in both children and adults. Furthermore, due to substantial indirect protection, the targeting of children is even predicted to reduce RSV in the elderly more than directly vaccinating the elderly themselves. Our results can help inform ongoing clinical trials and future recommendations on RSV vaccination.

Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells., Author Summary Single cell variation is of major interest in understanding key biological processes, like cancer, development and host pathogen interaction. During viral infection, these cell to cell variations can change the outcome of the whole organism infection. We suggested that differences in the number of parental viral genomes that initiate the replication process alter the outcome of infection among single cells. In this work we present a method based on genetically barcoded herpesvirus recombinants to identify the number of viral genomes initiating replication in individual cells. Our results indicate that viral gene expression is tightly linked to the number of viral genomes replicating per cell. Remarkably, we found that high cellular gene expression was an indicator for a lower viral gene expression in a given cell. We suggest that variations among single cells result from preexisting differences among cells, as well as from random viral host interactions.

Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments., IMPORTANCE Herpesviruses use nuclear factors and architecture to replicate their DNA genomes in the host nuclei. Viral replication compartments are distinct nuclear foci that appear during productive infection. We have recently developed a method that uses three viral recombinants (each expressing a different fluorescent protein) to quantify the number of incoming viral genomes that are expressed and replicated in each cell. We found that fewer than seven herpesvirus genomes can be expressed and replicated. Here we have expanded and improved upon our method and demonstrated that the phenomenon of limited genome expression is independent of the recombinants used. We correlated the small number of genomes expressed to the limited number of replication compartments by demonstrating that most replication compartments originate with a single genome. The distinction among replication compartments is maintained even when most of the nucleus is filled with viral DNA, implying that nuclear architecture constrains the compartments.

Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.