Human Cytomegalovirus TRS1 Protein Is Required for Efficient Assembly of DNA-Containing Capsids

Abstract

The human cytomegalovirus tegument protein, pTRS1, appears to function at several discrete stages of the virus replication cycle. We previously demonstrated that pTRS1 acts during the late phase of infection to facilitate the production of infectious virions. We now have more precisely identified the late pTRS1 function by further study of a mutant virus lacking the TRS1 region, ADsubTRS1. We observed a significant reduction in the production of capsids, especially DNA-containing C-capsids, in mutant virus-infected cells. ADsubTRS1 exhibited normal cleavage of DNA concatemers, so the defect in C-capsid production must occur after DNA cleavage and before DNA is stably inserted into a capsid. Further, the normal virus-induced morphological reorganization of the nucleus did not occur after infection with the pTRS1-deficient mutant.Cytomegalovirus, a member of the betaherpesvirus subfamily, is a large DNA virus with a narrow host range (19). Human cytomegalovirus (HCMV) infections are widespread and generally asymptomatic in healthy individuals. After infection, the virus can persist in a lifelong, latent state. Although relatively little is known about the events involved the establishment of HCMV latency, the cascade of gene expression and the events that occur during the lytic replication cycle of the virus have been studied in considerable detail (24).The genome of HCMV contains two unique domains, one long and one short, each flanked by repeat sequences. Two open reading frames, TRS1 and IRS1, include sequence from both repeated and unique domains. The N-terminal two-thirds of pTRS1 is encoded in the c repeat region, and the remainder of the protein is coded within the unique short region. The related protein, pIRS1, is encoded in the internal or cЈ repeat region plus the adjacent unique short region. Consequently, the N-terminal domains of pTRS1 and pIRS1 are nearly identical, and the two proteins have different C-terminal domains (37). Because their amino-terminal domains are encoded in the repeat region, the transcription of these genes is controlled by identical immediate-early promoters. Both pTRS1 and pIRS1 are packaged into the virion and therefore are delivered to the cell immediately upon infection (32); pTRS1 and pIRS1 can be detected in infected cells as early as 2 h postinfection (33, 36).The first function ascribed to pTRS1 and pIRS1 was transcriptional activation. Both proteins were found to act in conjunction with the immediate-early transcriptional regulatory proteins, IE1 and IE2, but not on their own, to increase expression from the UL44 promoter in transient-transfection assays (36). Subsequent analysis identified TRS1/IRS1 as 1 of 11 loci that are required for transient complementation of HCMV oriLyt-dependent DNA replication (28). In this assay, pTRS1 and pIRS1 likely facilitate the accumulation of other proteins that function directly in the replication process (15). More recently, pTRS1 and pIRS1 have been shown to block the double-stranded RNA-dependent protein kinase R response pathway,...

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

“…It has been reported to stimulate the expression of reporter genes driven by various HCMV promoters and to function in oriLyt-dependent DNA replication, both effects being observed in transient-expression assays (52,53,61,63). Additionally, TRS1 has been shown to play a role in viral capsid assembly (1), and deletion of the majority of the TRS1 open-reading frame from HCMV results in a growth defect that is most pronounced at a low MOI (8). IRS1, the TRS1 homologue in HCMV, also stimulates reporter gene expression but is not required for efficient replication of HCMV (8,31,42,61,63).…”

Tegument Proteins of Human Cytomegalovirus

“…No defects in viral mRNA accumulation were observed (3), perhaps because of the presence of the IRS1 gene in the TRS1 mutant. However, the absence of TRS1 resulted in an altered pattern of viral DNA accumulation in the nucleus and a defect in DNA packaging (3). As this is not observed in the absence of IRS1, this assembly function of TRS1 likely maps to the unique C terminus of the protein.…”

Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

“…Although pTRS1 was initially described as a transcriptional activator that stimulates gene expression in conjunction with the immediate-early proteins IE1p72 and IE2p86 (45), analysis of a TRS1-deleted virus revealed no delay in the accumulation of viral RNAs and proteins but did reveal a major defect at late times of the replicative cycle, which was further narrowed down to a role for pTRS1 in the assembly of DNA-containing capsids (1). However, the kinetics of viral DNA release in the supernatant of ⌬UL26-infected cells argue against an involvement of pUL26 in either capsid assembly or the loading of DNA into capsids.…”

“…Another tegument protein with recently proven importance for the efficient assembly of viral particles is pTRS1 (1,11). Although pTRS1 was initially described as a transcriptional activator that stimulates gene expression in conjunction with the immediate-early proteins IE1p72 and IE2p86 (45), analysis of a TRS1-deleted virus revealed no delay in the accumulation of viral RNAs and proteins but did reveal a major defect at late times of the replicative cycle, which was further narrowed down to a role for pTRS1 in the assembly of DNA-containing capsids (1).…”