Analysis of the early steps of herpes simplex virus 1 capsid tegumentation.

Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay. As anticipated, in vitro cytoplasmic capsids did not harbor UL34, UL31, or viral glycoproteins but contained US3. In agreement with previous findings, both nuclear and in vitro capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced in vitro without any cytosolic viral proteins also scored positive for UL36 and UL37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the in vitro assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.

Analysis of herpes simplex virus type I nuclear particles by flow cytometry.

Flow cytometry has been instrumental to characterize cell populations and examine their inner molecules and processes. In most instances, whole cells are analyzed, and hence, particle size is not an issue. Viruses are 2-3 orders of magnitude smaller than cells so flow cytometry has typically been used to study viral markers within whole infected cells. However, the ability to separate and purify viral particles representing different maturation stages within a viral life cycle would be a useful tool to analyze them in details and characterize the host proteins they associate with. Herpes simplex virus Type 1 is a 250 nm enveloped DNA virus that replicates in the nucleus where it assembles new viral particles called capsids. These capsids eventually travel to the cell surface and are modified along the way, producing several intermediate particles. In the nucleus, three types of stable nonenveloped 125 nm nuclear capsids exist that differ in protein composition and genome content. This includes so-called nuclear C-capsids that are the precursors of mature extracellular virions. We report that we can apply flow cytometry to sort these nuclear C-capsid intermediates by labeling the viral genome with Syto 13, a fluorescent marker that binds to nucleic acids. This is the first time flow cytometry has been used not only to detect but also to purify an intracellular viral maturation intermediate. This opens new research avenues in virology to study capsid assembly, maturation and egress, analyze mutant phenotypes, and define host factors associated with specific viral intermediates.

Biochemical analysis of infected cell polypeptide (ICP)0, ICP4, UL7 and UL23 incorporated into extracellular herpes simplex virus type 1 virions.

Herpes simplex virus type 1 (HSV-1) capsids assemble in the nucleus but acquire their teguments from various cellular compartments. Unfortunately, little is known about their exact arrangement and when they coat the newly produced capsids. The complexity of the virions is further highlighted by our recent proteomics analysis that detected the presence of several novel or controversial components in extracellular HSV-1 virions. The present study probes the localization and linkage to the virus particles of some of these incorporated proteins. We confirm the recently reported tight association of infected cell polypeptide (ICP)0 with the capsid and show that this property extends to ICP4. We also confirm our proteomics data and show biochemically that UL7 and UL23 are indeed mature virion tegument components that, unlike ICP0 and ICP4, are salt-extractable. Interestingly, treatment with N-ethylmaleimide, which covalently modifies reduced cysteines, strongly prevented the release of UL7 and UL23 by salts, but did not perturb the interactions of ICP0 and ICP4 with the virus particles. This hitheir at distinct biochemical properties of the virion constituents and the selective implication of reduced cysteines in their organization and dynamics. Finally, the data revealed, by two independent means, the presence of ICP0 and ICP4 on intranuclear capsids, consistent with the possibility that they may at least partially be recruited to the virus particles early on. These findings add significantly to our understanding of HSV-1 virion assembly and to the debate about the incorporation of ICP0 and ICP4 in virus particles.

Comprehensive characterization of extracellular herpes simplex virus type 1 virions.

The herpes simplex virus type 1 (HSV-1) genome is contained in a capsid wrapped by a complex tegument layer and an external envelope. The poorly defined tegument plays a critical role throughout the viral life cycle, including delivery of capsids to the nucleus, viral gene expression, capsid egress, and acquisition of the viral envelope. Current data suggest tegumentation is a dynamic and sequential process that starts in the nucleus and continues in the cytoplasm. Over two dozen proteins are assumed to be or are known to ultimately be added to virions as tegument, but its precise composition is currently unknown. Moreover, a comprehensive analysis of all proteins found in HSV-1 virions is still lacking. To better understand the implication of the tegument and host proteins incorporated into the virions, highly purified mature extracellular viruses were analyzed by mass spectrometry. The method proved accurate (95%) and sensitive and hinted at 8 different viral capsid proteins, 13 viral glycoproteins, and 23 potential viral teguments. Interestingly, four novel virion components were identified (U(L)7, U(L)23, U(L)50, and U(L)55), and two teguments were confirmed (ICP0 and ICP4). In contrast, U(L)4, U(L)24, the U(L)31/U(L)34 complex, and the viral U(L)15/U(L)28/U(L)33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of U(L)23. Surprisingly, the viral glycoproteins gJ, gK, gN, and U(L)43 were absent. Analyses of virions produced by two unrelated cell lines suggest their protein compositions are largely cell type independent. Finally, but not least, up to 49 distinct host proteins were identified in the virions.