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. FASEB J. 33, 9388–9403 (2019). http://www.fasebj.org

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, Valporic Acid, and Suberoylanilide Hydroxamic Acid. We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the gene expression from the viral genomes. 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 (promyelocytic leukemia and ATRX), which may explain the lower 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.

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.

21Homologous recombination (HR) is considered a major driving force of evolution since 22 it generates and expands genetic diversity. Evidence of HR between co-infecting 23 herpesvirus DNA genomes can be found frequently, both in vitro and in clinical isolates. 24 Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from 25 a single incoming genome and maintains a specific territory within the nucleus. This 26 raises intriguing questions about where and when co-infecting viral genomes interact. 27 To study the spatiotemporal requirements for inter-genomic recombination, we 28 developed an assay with dual-color fluorescence in situ hybridization which enables 29 detection of HR between different pairs of co-infecting HSV-1 genomes. Our results 30 revealed that when viral RCs enlarge towards each other, there is detectable overlap 31 between territories of genomes from each virus. Infection with paired viruses that allow 32 visualization of HR correlates with increased overlap of RCs. Further, inhibition of RC 33 movement reduces the rate of HR events among co-infecting viruses. Taken together, 34 these findings suggest that inter-genomic HR events take place during replication of 35 HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce. Our 36 observations have implications on understanding the recombination restrictions of 37 other DNA viruses and cellular DNA.38 39 40 41Recombination is considered to be a major driving force in evolution of most organisms, 42 since it accelerates adaptation (1,2). The architecture of eukaryotic nuclei is suggested 43 to regulate many DNA-mediated processes, including replication, gene expression 44 and recombination. DNA viruses that replicate inside the nucleus clearly change the 45 nuclear architecture, however they are subjected to similar spatial constraints as host 46 DNA. The rate of mutation accumulation is lower for DNA viruses than that of viruses 47 with RNA genomes (3,4). It has been hypothesised that high rates of recombination 48 can facilitate genetic adaptation to the changing environment (5). Indeed, homologous 49 recombination (HR) among co-infecting Herpes simplex virus type 1 (HSV-1) genomes 50 is very frequently observed in both in vitro genetic assays (6-12) and in sequence 51 analysis of clinical isolates (13-15). Herpesvirus infection therefore provide a system 52 to study spatial features that promote or constrain recombination in the eukaryotic 53 nucleus. 54 Like all other herpesviruses, HSV-1 viral gene expression, replication and capsid 55 assembly all occur in the host nucleus of infected cells. Viral genomes enter the 56 nucleus through the nuclear pore complex as naked DNA molecules (16), and these 57 rapidly recruit several host and viral proteins to the viral genomes (17-25). Expression 58 of the immediate early and early viral genes allows initiation of viral DNA replication 59 (26). HSV-1 DNA replication proceeds at distinct foci within the nucleus known as 60 replication compartments (RC...

Herpesviruses are highly prevalent and cause significant morbidity in the human and animal populations. Most individuals who are infected with herpes simplex virus (HSV-1), a common human pathogen, will become lifelong carriers of the virus, as HSV-1 establishes latent (quiescent) infections in the host cells.