Structures of the portal vertex reveal essential protein-protein interactions for Herpesvirus assembly and maturation

Dear Editor,Herpesviridae is a large family of double-stranded DNA(dsDNA)viruses that cause a variety of human diseases ranging from cold sores and chicken pox to congenital defects,blindness and cancer(Chayavichitsilp et al.,2009;Wang et al.,2018).In the past 70 years,substantial advances in our knowledge of the molecular biology of herpesviruses have led to insights into disease pathogenesis and management.However,the mechanism for capsid assembly that requires the ordered packing of about 4,000 protein subunits into the hexons,pentons and triplexes remains elusive.It is still a puzzle how initially identical subunits adopt both hexameric and pentameric conformations in the capsid and select the correct locations needed to form closed shells of the proper size.Biochemical and genetic studies have shown that the portal is involved in initiation of capsid assembly(Newcomb et al.,2005)and functions akin to a DNA-sensor coupling genome-packaging achieved by a genome-packaging machinery-“terminase complex”(Chen et al.,2020;Yunxiang Yang,2020)with icosahedral capsid maturation(Lokareddy et al.,2017).Structural investigations of the herpesvirus portal have proven challenging due to the small size of this dodecamer,which accounts for less than 1%of the total mass of the capsid protein layer and the technical difficulties involved in resolving non-icosahedral components of such large icosahedral viruses(diameter is∼1,250Å).Efforts of many investigators over two decades have made to reconstruct the cryo-electron microscopy(cryo-EM)structure of herpesvirus portal vertex and more recently near-atomic structures of two herpesvirus(herpes simplex virus type 1(HSV-1)and Kaposi’s sarcoma-associated herpesvirus(KSHV))portal vertices were reported(McElwee et al.,2018;Gong et al.,2019;Liu et al.,2019).

Termination of Transcription of LAT Increases the Amounts of ICP0 mRNA but Does Not Alter the Course of HSV-1 Infection in Latently Infected Murine Ganglia

On entering sensory ganglia,herpes simplex viruses 1(HSV-1)establishes a latent infection with the synthesis of a latency associated transcript(LAT)or initiates productive infection with expression of a set of immediate early viral proteins.The precise mechanisms how expression of a genes is suppressed during the latency are unknown.One mechanism that has been proposed is illustrated in the case of ICP0,a key immediate early viral regulatory protein.Specifically,the 2 kb LAT intron is complementary to the 30 terminal portion of ICP0 m RNA.To test the hypothesis that accumulation of LAT negatively affects the accumulation of ICP0 m RNA,we inserted a DNA fragment encoding two poly(A)sequences into LAT to early terminate LAT transcript without interrupting the complementary sequence of ICP0 transcript(named as SR1603).Comparisons of the parent(SR1601)and mutant(SR1603)HSV-1 viruses showed the following:Neurons harboring latent SR1603 virus accumulated equivalent amounts of viral DNA but higher amounts of ICP0 m RNA and lower amounts of LAT,when compared to neurons harboring the SR1601 virus.One notable difference between the two viruses is that viral RNA accumulation in explanted ganglia harboring SR1603 virus initiated significantly sooner than that in neurons harboring SR1601 virus,suggesting that ICP0 may act as an activator of viral gene expression in permissive cells.Collectively,these data suggest that increased ICP0 m RNA by suppressed LAT did not affect the establishment of latency in latently infected murine ganglia.

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication

To date, 29 distinct microRNAs(miRNAs) have been reported to be expressed during herpes simplex virus infections.Sequence analysis of mature herpes simplex virus-1(HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27.However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield.Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.