Abstract
ABSTRACTIt is now well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx, and ATRX while human cytomegalovirus (HCMV) IE protein IE1 targets PML and Sp100, and its tegument protein pp71 targets hDaxx and ATRX. The functions of these viral regulatory proteins are in part interchangeable; thus, both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by other investigators to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings reinforce the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections.IMPORTANCE Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen's DNA, while viral functions are expressed that counteract the cell-mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as PML nuclear bodies (PML NBs), or ND10. Members of the alpha-, beta-, and gammaherpesvirus families all express proteins that interact with, degrade, or otherwise counteract the inhibitory effects of various PML NB components. Previous work has shown that there is the potential for a functional interchange between the viral proteins expressed by alpha- and betaherpesviruses, despite a lack of obvious sequence similarity. Here, this concept is extended to include a member of the gammaherpesviruses.
Highlights
It is well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection
It was found that the functions of herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 could be at least partially replaced by members of the ICP0 family of proteins expressed by other alphaherpesviruses [11]
Recent work has shown that BNRF1, a member of the ORF75c family of proteins that are represented in the teguments of many gamma herpesviruses [5], is required for efficient Epstein-Barr virus (EBV) infection through a mechanism involving interaction with hDaxx and disruption of the hDaxx/ATRX complex [30, 31]
Summary
Lentivirus vector plasmids expressing the tetracycline repressor linked to a nuclear localization signal (NLS) and enhanced green fluorescent protein (pLKOneo.EGFPnlsTetR) and IE1 or myc-tagged pp from a tetracycline-inducible promoter have been described previously [22, 37]. For single inducible expression of these proteins, the BNRF1 vector and the puromycin resistance version of the EBNA-LP were used to transduce HA-TetR cells. For expression of EBNA-LP in HFT-derived cells, sequential transductions were performed with a polycistronic vector expressing puromycin resistance and the tetracycline repressor (and enhanced yellow fluorescent protein [EYFP] in an inducible manner [kindly provided by Chris Boutell]) with the G418-resistant version of the EBNA-LP-expressing vector. For determination of relative plaque-forming efficiencies of ICP0-null mutant HSV-1, cells were seeded for plaque assays into 24-well dishes at 1 ϫ 105 cells per well and infected the following day with appropriate sequential 3-fold dilutions of dl1403/ CMVlacZ. The images were exported as TIF files, minimally adjusted using Photoshop, and assembled into figures using Illustrator
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