Herpesvirus Tegument Protein pUL37 Interacts with Dystonin/BPAG1 To Promote Capsid Transport on Microtubules during Egress

To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication. IMPORTANCEHerpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment. Herpesviruses have a unique virion structure designated the tegument, which is a proteinaceous layer consisting of a number of different viral proteins and is located between the nucleocapsid and the envelope (1). Herpes simplex virus 1 (HSV-1), classified in the Alphaherpesvirinae subfamily of the Herpesviridae family, is one of the best-studied members in the family and has at least 23 different viral proteins in the tegument (2).In HSV-1-infected cells, packaging of nascent progeny...

Section: Discussionmentioning

confidence: 99%

The Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis

Oda

Arii

Koyanagi

et al. 2016

“…UL36p and UL37p can also associate with organelles in the absence of capsids, since they are assembled into L particles (enveloped structures lacking capsids) in a mutually interdependent fashion (47)(48)(49) and because UL36p can mediate capsid-independent targeting of UL37p to the TGN (50). Loss of UL37p and UL36p results in similar phenotypes: accumulation of nonenveloped capsids in the nucleus and cytosol (27,41,51,52) and a decrease in microtubule-mediated transport of capsids (39,40,42,53). The interplay between UL36p/UL37p, the ESCRT apparatus, and other virally encoded proteins during capsid-membrane association and envelopment (8,10,46,54) is not well understood.…”

mentioning

confidence: 99%

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

Kharkwal

Furgiuele

Smith

et al. 2015

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsidmembrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCEMembrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment. Herpesviruses replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is then generally accepted that capsids bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These naked cytoplasmic capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (4-9). Completion of cytoplasmic envelopment requires the coordinated interaction of multiple virally encoded proteins (8,(10)(11)(12)(13) and the participation of components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery (14-19).UL36p (VP1/2) is the largest structural protein encoded by the herpesviridae (20, 21). It is a major constituent of the innermost layer of tegument, the complex protein layer between the capsid and the inner surface of the envelope, and connects the capsid to m...

“…Interestingly, HSV-1 pUL37 has recently been shown to interact with the MT-binding protein dystonin/BPAG1 to promote capsid transport on microtubules (52). Moreover, the same group showed that dystonin silencing during entry impaired HSV-1 capsid accumulation as a ring around the nuclear periphery (53), a phenotype very similar to the one observed for the MuHV-4 ORF63 STOP mutant (Fig.…”

Section: Discussionmentioning

confidence: 68%

Deletion of Murid Herpesvirus 4 ORF63 Affects the Trafficking of Incoming Capsids toward the Nucleus

Latif

Machiels

Xiao

et al. 2016

Gammaherpesviruses are important human and animal pathogens. Despite the fact that they display the classical architecture of herpesviruses, the function of most of their structural proteins is still poorly defined. This is especially true for tegument proteins. Interestingly, a potential role in immune evasion has recently been proposed for the tegument protein encoded by Kaposi's sarcoma-associated herpesvirus open reading frame 63 (ORF63). To gain insight about the roles of ORF63 in the life cycle of a gammaherpesvirus, we generated null mutations in the ORF63 gene of murid herpesvirus 4 (MuHV-4). We showed that disruption of ORF63 was associated with a severe MuHV-4 growth deficit both in vitro and in vivo. The latter deficit was mainly associated with a defect of replication in the lung but did not affect the establishment of latency in the spleen. From a functional point of view, inhibition of caspase-1 or the inflammasome did not restore the growth of the ORF63-deficient mutant, suggesting that the observed deficit was not associated with the immune evasion mechanism identified previously. Moreover, this growth deficit was also not associated with a defect in virion egress from the infected cells. In contrast, it appeared that MuHV-4 ORF63-deficient mutants failed to address most of their capsids to the nucleus during entry into the host cell, suggesting that ORF63 plays a role in capsid movement. In the future, ORF63 could therefore be considered a target to block gammaherpesvirus infection at a very early stage of the infection. IMPORTANCEThe important diseases caused by gammaherpesviruses in human and animal populations justify a better understanding of their life cycle. In particular, the role of most of their tegument proteins is still largely unknown. In this study, we used murid herpesvirus 4, a gammaherpesvirus infecting mice, to decipher the role of the protein encoded by the viral ORF63 gene. We showed that the absence of this protein is associated with a severe growth deficit both in vitro and in vivo that was mainly due to impaired migration of viral capsids toward the nucleus during entry. Together, our results provide new insights about the life cycle of gammaherpesviruses and could allow the development of new antiviral strategies aimed at blocking gammaherpesvirus infection at the very early stages. G ammaherpesviruses (␥HVs) are widespread viruses that cause lifelong infections in many mammalian species (1) and represent a significant cause of diseases (2, 3). Epstein-Barr virus (EBV; genus Lymphocryptovirus) and Kaposi's sarcoma-associated herpesvirus (KSHV; genus Rhadinovirus genus) are highly prevalent in human populations (3, 4) and are associated with several cancers (5). Despite the burden associated with these infections in some regions of the world, there is still no standard treatment (6). A better understanding of their biological cycle is therefore needed to develop new prophylactic or therapeutic strategies. As EBV and KSHV replicate poorly in vitro and have no establi...