Analyse des protéines du tégument par virométrie en flux et protéomique des capsides nucléaires du Virus Herpès Simplex de type 1 (VHS-1)

Herpes simplex virus type 1 (HSV-1) is a highly contagious infectious agent that constitutes a real global health problem and can cause many pathologies ranging from cold sores to encephalitis and systemic disease in newborns. The virus is composed of 4 distinct structural components: a linear double-stranded DNA viral genome encompassed within a 125 nm icosahedral capsid, a layer of protein called the tegument, and a host-derived lipid envelope in which the viral glycoproteins are anchored. The tegument is a very dense and very complex network comprising thousands of proteins of different sizes that structurally bridge the viral envelope to the capsid. These multifunctional proteins perform several important functions throughout the viral cycle. They are involved in transport and targeting of incoming capsids to nuclear pores, maturation and egress of newly made viral particles, and the acquisition of the final envelope. Tegument proteins have been extensively studied to determine their roles in infection and virulence. However, being limited by conventional techniques, all these studies consider the entire viral population to be responsible for the infectious phenotype without considering the possible heterogeneity of specific tegument proteins among viral particles and its effect on infectivity. Following fusion of the viral envelope with the plasma membrane leading to entry of the virus, the viral genome is delivered to the nucleus, where it replicates and leads to the assembly of new capsids. Here, four distinct nonenveloped capsid species are present. The thermodynamically unstable procapsids, the A‐capsids that fail to properly incorporate the viral genome, the B‐capsids that also lack viral DNA and the C‐capsids that incorporate the viral genome and ultimately form mature enveloped virions. Those capsids can be distinguished from each other based on their composition (DNA and proteins) contents and their appearance in electron microscopy. A- and B-capsids are considered to be abortive and only mature C-capsids can travel across the two nuclear membranes by an envelopment/de‐envelopment mechanism and are ultimately re‐enveloped in the TGN. The acquisition of the tegument layer is believed to be sequential from the nucleus to the TGN via the cytoplasm. This acquisition is favored by a very complex network of protein interactions involving the capsid and the proteins of the tegument. However, the exact sequence of addition of these proteins is still poorly defined. The first paper presented in this thesis has been published in Journal of Virology. Here, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach we developed in the laboratory. Our data confirm that while some viral proteins are incorporated in controlled amounts, others vary substantially. We also highlighted the correlation between the abundance of specific tegument proteins and the infectivity of the virions. In the second paper, the use of flow virometry enabled us not only to analyze the nuclear capsids, but also to increase the purity of C-capsids. And for the first time, we were able to analyze three types of nuclear capsids (A, B and C) by mass spectrometry and determine their overall protein composition. These observations strongly support the hypothesis that acquisition of the tegument proteins starts early at the nucleus and support the likely involvement of these proteins in the primary envelopment of the capsids. We also noted the presence of host proteins associated with capsids.

Keyphrases: HSV-1, Nuclear capsids, Flow cytometry, Flow virometry, Mass spectrometry, Proteomics, Infectivity