Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy

Liashkovich, Ivan; Hafezi, Wali; Kühn, Joachim; Oberleithner, Hans; Kramer, Armin; Shahin, Victor

doi:10.1242/jcs.032284

Cited by 44 publications

(37 citation statements)

References 28 publications

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“…HSV capsids are known to undergo structural changes as they mature from procapsid to C capsid during the encapsidation reaction and as they further develop into extracellular virions (34,54,66). In this report, we demonstrated that capsids lacking disulfide bonds are unstable and prone to the loss of pentons and peripentonal triplexes.…”

Section: Discussionmentioning

confidence: 71%

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Szczepaniak

Nellissery

Jadwin

et al. 2011

J Virol

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Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent crosslinks, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.Virus capsids have evolved mechanisms to protect viral genomes from the extracellular environment while maintaining the ability to release the viral genome upon entry into a new host cell during the next round of infection. The formation of capsids containing the herpes simplex virus type 1 (HSV-1) double-stranded DNA (dsDNA) genome is a complex process involving a preassembled protein shell (procapsid) containing the viral protease and scaffolding proteins (encoded by UL26 and UL26.5), concatemeric viral DNA, and seven cleavage and packaging proteins (4,13,24). The capsid shell is composed primarily of the major capsid protein (VP5), two triplex proteins (VP19C and VP23), and VP26, as well as a dodecameric UL6 portal ring located at a unique vertex through which DNA most likely is packaged and released.During wild-type (WT) infection, three capsid forms are observed: A capsids, which have participated in an abortive encapsidation process and have lost both scaffold proteins and DNA; B capsids, which contain proteolytically processed forms of the internal scaffold proteins but no DNA; and DNA-containing mature C capsids, which have lost scaffold during the process of taking up DNA (18). DNA-containing C capsids undergo structural alterations which result in the acquisition of increased amounts of a heterodimer made up of UL25 and UL17 (81) believed to stabilize DNA-containing capsids (10,12...

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Section: Discussionmentioning

confidence: 71%

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Szczepaniak

Nellissery

Jadwin

et al. 2011

J Virol

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“…Thus, a 5-fold difference in the concentration of monovalent salts did not influence the capsid rigidity in our system. Whereas we studied the mechanical properties of nuclear capsids, Liashkovich et al (17) analyzed viral capsids that had been isolated from viral particles secreted from infected cells, and thus most likely contained significant amounts of tegument attached to them (45,46). The tegument may well increase the particle spring constant, as the latter is proportional to the square of the shell thickness (see Eq.…”

Section: Discussionmentioning

confidence: 99%

“…B, A, and C capsids all have mature angularized shells, but biochemical and electron microscopy experiments show that there are differences in their protein composition and their morphology (8, 16). However, it is unclear whether they also differ in terms of mechanical properties (16,17). Recently developed nanoindentation techniques using atomic force microscopy (AFM) allow for the analysis of mechanical properties of viruses at the single particle level (18)(19)(20).…”

mentioning

confidence: 99%

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Roos

Radtke

Kniesmeijer

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

122

123

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Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA.atomic force microscopy ͉ nanoindentation ͉ penton ͉ viral structure ͉ virus mechanics H erpes simplex virus type 1 (HSV1) virions encapsidate their 152 kbp double-stranded DNA genome in an icosahedral capsid that is surrounded by an amorphous protein layer, called the tegument, and a lipid-containing envelope. Assembly of herpesviruses is initiated in the nucleus where procapsids selfassemble around a protein scaffold and subsequently mature (1-4). This transformation is characterized by massive conformational changes of the Ϸ200 MDa shell, resulting in stable, mature capsids, and is in many aspects analogous to maturation processes in bacteriophages (5, 6). The scaffold is proteolytically cleaved and removed, whereas the outer shell transforms from a spherical into an icosahedral shape (7, 8). The mature capsid has an outer diameter of 125 nm with an overall shell thickness of Ϸ15 nm (9). Capsomeres converge at their proximal ends, forming a contiguous shell of Ϸ4 nm thickness that is only interrupted by channels passing through all of the 162 capsomeres (10, 11). The capsomeres are hexamers (hexons) and pentamers (pentons) of the major capsid protein VP5 that form a shell with a triangulation number of T ϭ 16. In total, 320 triplexes, each formed by a heterotrimer of 1 VP19c and 2 VP23 molecules, connect these capsomeres (10). One of the 12 capsid vertices is occupied by the pUL6 portal (12). Three particle types can be isolated from the nuclei of infected cells due to their different sedimentation behavior: B capsids, which still have the scaffold inside; the lighter A capsids, which are empty; and the denser C capsids, which contain the DN...

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“…The DNA-anisotropic mechanical reinforcement observed in the MVM , lead to the proposition that the lower rigidity measured at the fivefold axes where the capsid pores are located, could possibly have an adaptive biological role useful for posterior infectivity. The exceptional mechanical stability found for the HSV-1 virus might be a key factor for the survival during transport over long distances of the axonal cytoplasm where is exposed to mechanical stresses by molecular motors before it reaches its final point for cargo release (Liashkovich, et al, 2008). The reported mechanical stability and self-healing mechanism in Norovirus capsids (Cuellar, et al, 2010) with regard to drastic changes in pH may be an essential requirement for infection.…”

Section: Viral Mechanics and The Infection Cyclementioning

confidence: 97%

“…The envelope-free capsid could withstand maximal compression forces of about 6 nN (Liashkovich, et al,2008). Mechanical failure of the DNA containing capsids was observed at loading forces of about 9 nN.…”

mentioning

confidence: 98%

Force Microscopy – A Tool to Elucidate the Relationship Between Nanomechanics and Function in Viruses

Cuellar¹,

Donath²

2012

Atomic Force Microscopy Investigations Into Biology - From Cell to Protein

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Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy

Cited by 44 publications

References 28 publications

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Force Microscopy – A Tool to Elucidate the Relationship Between Nanomechanics and Function in Viruses

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