Promoting Simultaneous Onset of Viral Gene Expression Among Cells Infected with Herpes Simplex Virus-1

Abstract

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.

Mutational pressure by host APOBEC3s more strongly affects genes expressed early in the lytic phase of herpes simplex virus-1 (HSV-1) and human polyomavirus (HPyV) infection

“…In vitro experiments have previously shown that HSV-1 infection at differing MOI yields differential viral gene transcription rates [ 26 , 47 ]. Using our model, we sought to evaluate whether the virus can mitigate the effects of genome mutations by infecting at a higher MOI.…”

Cellular and Viral Determinants of HSV-1 Entry and Intracellular Transport towards Nucleus of Infected Cells

“…The major capsid protein VP5 (ICP5) and the inner tegument protein UL37 recruit the dynein motor and other accessory proteins such as BICD2, EB1, and the dynactin complex (DCTN) to facilitate retrograde transport of virion capsids. AKt and PKC activities are required for efficient entry and transport along the microtubules towards the nucleus, where the virus replicates its DNA (Adapted from Ralph et al ., 2017, (89)).…”

The Fate of Incoming HSV-1 Genomes Entering the Nucleus

“…These three papers (Sekine et al, 2017;Alandijany et al, 2018;Dembowski and DeLuca, 2018) used three different methods to initiate infection and determine the initial time of infection. This emphasizes the need for an accepted synchronization method for recognizing initial events of incoming viral genomes (Ralph et al, 2017).…”

“…Currently, one of the best way to synchronize nuclear entry is using the tsB7 mutant (or its derivatives), which permits the capsids to bind the nuclear pore complex at high temperature but allows rapid injection of viral genome into the nucleus only at low temperature (Batterson et al, 1983;Abaitua et al, 2009;Abaitua et al, 2011;Ralph et al, 2017). However, these temperature changes might affect other viral or host processes (Ralph et al, 2017). Alternative approaches for synchronizing nuclear entry are needed.…”