Proteomics of Herpes Simplex Virus 1 Nuclear Capsids

Abstract

Herpes simplex virus replicates in the nucleus, where new capsids are assembled. It produces procapsids devoid of nucleic acid but containing the preVP22a scaffold protein. These thermo-unstable particles then mature into A-, B- or C-nuclear icosahedral capsids, depending on their ability to shed the proteolytically processed scaffold and incorporation of the viral genome. To study how these viral capsids differ, we performed proteomics studies of highly enriched HSV-1 A-, B- and C-nuclear capsids, relying in part on a novel and powerful flow virometry approach to purify C-capsids. We found that the viral particles contained the expected capsid components and identified several tegument proteins in the C-capsid fraction (pUL21, pUL36, pUL46, pUL48, pUL49, pUL50, pUL51 and pUS10). Moreover, numerous ribosomal, hnRNPs and other host proteins, absent from the uninfected controls, were detected on the capsids with some of them seemingly specific to C-capsids (glycogen synthase, four different keratin-related proteins, fibronectin 1 and PCBP1). A subsequent proteomics analysis was performed to rule out the presence of protein complexes that may share similar density as the viral capsids but do not otherwise interact with them. Using pUL25 or VP5 mutant viruses incapable of assembling C-nuclear or all nuclear capsids, respectively, we confirmed the bulk of our initial findings. Naturally, it will next be important to address the functional relevance of these proteins. IMPORTANCE Much is known about the biology of herpesviruses. This includes their unique ability to traverse the two nuclear envelopes by sequential budding and fusion steps. For HSV-1, this implies the pUL31/pUL34 and pUL17/pUL25 complexes that may favor C-capsid egress. However, this selection process is not clear, nor are all the differences that distinguish A-, B- and C-capsids. The present study probes what proteins compose these capsids, including host proteins. This should open up new research avenues to clarify the biology of this most interesting family of viruses. It also reiterates the use of flow virometry as an innovative tool to purify viral particles.

The herpes simplex virus tegument protein pUL21 is required for viral genome retention within capsids

“…The capsid-associated tegument protein pUL21 is one of several viral proteins that function in nuclear egress and is conserved amongst the alphaherpesvirus subfamily [29][30][31][32]. pUL21 forms a tripartite complex in the mature HSV virion, interacting with tegument protein pUL16, which in turn interacts with pUL11 [33][34][35][36][37]. During the early stages of infection, pUL21 has been implicated in the trafficking of capsids to nuclei in HSV-1, HSV-2, and pseudorabies virus (PRV) infected cells as well as being required for optimal viral gene expression [31,[38][39][40].…”

Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly

“…The HSV-1 capsid assembly process offers a unique opportunity to accomplish this due to the ability to isolate B- and A-capsids, the dead-end products of C-capsid assembly. As mentioned above, while B- and A-capsids are not considered to be assembly intermediates because they are stably copurified with C-capsids, they directly reflect the intermediate states of capsid maturation toward C-capsid with associated DNA packaging progression ( 13 , 46 – 48 ). High-resolution cryo-electron microscopy and tomography experiments conducted on each stable form of herpesvirus particles ( 29 , 33 , 44 , 49 – 52 ) yielded 3D particle reconstructions that highlighted the structural distinctions between procapsids, B-, A-, and C-capsids, and enveloped virions.…”

Motor Skills: Recruitment of Kinesins, Myosins and Dynein during Assembly and Egress of Alphaherpesviruses

“…UL36p and UL37p perform multiple functions including capsid envelopment in the cytoplasm [ 12 , 17 , 20 , 30 , 109 ], recruitment of outer tegument polypeptides [ 12 , 13 , 110 , 111 ] and, as detailed in Section 3.2 , Section 3.3 , Section 3.4 and Section 3.5 , motor recruitment. UL36p and UL37p become attached to alphaherpesvirus capsids in the nucleoplasm or soon after their entry into the cytoplasm ([ 112 , 113 , 114 , 115 , 116 , 117 , 118 ], recently reviewed in [ 17 ]). This ensures that, immediately upon their export from the nucleus, capsids are poised to recruit kinesin motors and begin their journey through the cytoplasm [ 12 , 21 ].…”