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

Abstract

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...

Helminth-induced IL-4 expands bystander memory CD8+ T cells for early control of viral infection

“…Viral genome loads were measured using a Taqman-based real-time PCR reaction (CFX) 56 . DNA was extracted from spleens using Wizard genomic DNA purification kit (Promega) and 100 ng per sample was used in iQ supermix (Biorad) to amplify a MuHV-4-specific sequence (genomic co-ordinates 43,015–43,138, NC_001826.2): gene ORF25 with primers ORF25fwd, 5′-atggtatagccgcctttgtg-3′ and ORF25rev 5′-acaagtggatgaagggttgc-3′ and probe 5′-6-carboxyfluorescein [FAM]-caaccactggatcagcataaaacttatgaa-black hole quencher [BHQ1]-3′.…”

In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4

“…Thus, phenotypes observed previously with v-kLANA ( 24 ) or here with C-terminal swap viruses are not due to altered M11 expression. In addition, M3 and ORF63 are lytic genes that encode a chemokine binding protein ( 10 , 37 , 38 ) and a tegument protein ( 39 , 40 ), respectively. Neither M3 nor ORF63 mRNA levels were reduced in v-kLANA.yfp or v-KM.yfp compared to v-WT.yfp ( Fig.…”

Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68

“…Similarly, ORF49 protein, a derepressor of RTA, is required for efficient lytic replication both in vitro and in vivo (15). Recent studies validated critical roles in the acute phase for lytic genes ORF35 (16), ORF48 (17), and ORF63 (18), in addition to genetic elements including the lytic origins of replication (oriLyt) (19). Viral gene products essential for key facets of the acute phase, such as cell type-specific infectivity or immune evasion, cannot be accurately modeled in cell culture.…”

“…Similarly, ORF49 protein, a derepressor of RTA, is required for efficient lytic replication both in vitro and in vivo ( 15 ). Recent studies validated critical roles in the acute phase for lytic genes ORF35 ( 16 ), ORF48 ( 17 ), and ORF63 ( 18 ), in addition to genetic elements including the lytic origins of replication (oriLyt) ( 19 ).…”