Phosphorylation of E2 Serine Residue 402 Is Required for the Transcription and Replication of the HPV5 Genome.

Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic ß-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many ß-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of ß-HPV5 replication and its regulation by host cell protein kinases.

Sulfolobus islandicus Employs Orc1-2-Mediated DNA Damage Response in Defense against Infection by SSV2.

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

Betaherpesvirus assembly and egress: Recent advances illuminate the path.

The human betaherpesviruses, human cytomegalovirus (HCMV; species Human betaherpesvirus 5) and human herpesviruses 6A, 6B, and 7 (HHV-6A, -6B, and -7; species Human betaherpesviruses 6A, 6B, and 7) are highly prevalent and can cause severe disease in immune-compromised and immune-naive populations in well- and under-developed communities. Herpesvirus virion assembly is an intricate process that requires viral orchestration of host systems. In this review, we describe recent advances in some of the many cellular events relevant to assembly and egress of betaherpesvirus virions. These include modifications of host metabolic, immune, and autophagic/recycling systems. In addition, we discuss unique aspects of betaherpesvirus virion structure, virion assembly, and the cellular pathways employed during virion egress.