The Terminase Complex of Each Human Herpesvirus

Abstract

The human herpesviruses (HHVs) are classified into the following three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. These HHVs have distinct pathological features, while containing a highly conserved viral replication pathway. Among HHVs, the basic viral particle structure and the sequential processes of viral replication are nearly identical. In particular, the capsid formation mechanism has been proposed to be highly similar among herpesviruses, because the viral capsid-organizing proteins are highly conserved at the structural and functional levels. Herpesviruses form capsids containing the viral genome in the nucleus of infected cells during the lytic phase, and release infectious virus (i.e., virions) to the cell exterior. In the capsid formation process, a single-unit-length viral genome is encapsidated into a preformed capsid. The single-unit-length viral genome is produced by cleavage from a viral genome precursor in which multiple unit-length viral genomes are tandemly linked. This encapsidation and cleavage is carried out by the terminase complex, which is composed of viral proteins. Since the terminase complex-mediated encapsidation and cleavage is a virus-specific mechanism that does not exist in humans, it may be an excellent inhibitory target for anti-viral drugs with high virus specificity. This review provides an overview of the functions of the terminase complexes of HHVs.

1. INTRODUCTION

Herpesviruses are comprised of an icosahedral capsid containing double-stranded DNA, a tegument composed of viral proteins, and a lipid envelope derived from host cells. In the family Herpesviridae, nine HHVs have been identified: herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), human herpesvirus 6B (HHV-6B), human herpesvirus 7 (HHV-7), and Kaposi’s sarcoma-associated herpesvirus (KSHV). They are further divided into three subfamilies (α-, β-, γ-) based on their biological characteristics. The α-herpesvirinae subfamily includes HSV-1, HSV-2, and VZV; the β-herpesvirinae subfamily includes HCMV, HHV-6A, HHV-6B, and HHV-7; and the γ-herpesvirinae subfamily includes EBV and KSHV. These HHVs establish a life-long infection in the host and exist as either a latent phase or a lytic phase (i.e., viral replication stage). In the initial infection and the lytic phase, herpesviruses cause various diseases in infected individuals. After the initial infection, these viruses maintain a latent state in healthy individuals. The infection state is switched from the latent phase to the lytic phase by viral reactivation. The lytic phase is induced under conditions of UV exposure, immunodeficiency, drug administration, or hormonal changes.

The progeny virus that possesses the ability to infect a new host is referred to as a virion. Virion production in herpesviruses occurs during the lytic phase after viral reactivation (Fig. 1). In this process, many viral lytic-related proteins are expressed. Furthermore, during the lytic phase, a viral genome precursor, in which multiple units of the viral genome are tandemly linked, is replicated, and a capsid forms through the self-assembly of major capsid proteins and scaffold proteins. Next, a single unit-length viral genome is cleaved from the genome precursor and is packaged into a capsid within the infected cell nucleus. After the mature capsid egresses from the nucleus and enters the cytoplasm, the capsid acquires the tegument (which is composed of viral tegument proteins), and the envelope, which is derived from the lipid bilayer of a secretory vesicle from the Golgi apparatus (or endoplasmic reticulum). Subsequently, this mature viral particle buds out of the cell as a virion. Although there are many processes involved in virion production, current evidence suggests that the rate-limiting step is the cleavage of the single-unit-length genome from the viral genome precursor and its packaging into the capsid. The cleavage and packaging steps require the viral terminase complex, which cleaves the DNA regions that contain repeating sequences referred to as terminal repeats (TRs). TRs are located in the linker DNA regions that connect each genome unit of a viral genome precursor. The TR cleavage mechanism by the terminase complex is essential for capsid maturation and is highly conserved among all herpesviruses. Since this mechanism is virus-specific and does not exist in humans, it may be a useful target for inhibition by anti-viral drugs. This review focuses on the packaging and cleavage mechanism and outlines the respective terminase complexes of HHVs.

Fig. 1. Mechanism of Herpesvirus Production in the Lytic State

Herpesviruses express viral lytic proteins during the lytic phase. Viral capsids are then formed within the infected cell nucleus. During viral genome replication, the viral genome is replicated as a genome precursor, in which the single-unit-length viral genomes are tandemly linked via linker DNA containing the terminal repeats (TRs). The herpesvirus terminase complex packages the single-unit-length viral genome into a preformed capsid and cleaves the viral genome precursor at the TR site. Subsequently, the mature capsid containing the viral genome egresses from the nucleus and translocates to the cytoplasm. In the cytoplasm, the mature capsid acquires tegument proteins as well as an envelope, and buds out of the cell as a virion.

2. THE MECHANISM OF HERPESVIRUS CAPSID MATURATION

The capsid maturation process and its mechanisms are better understood for HSV-1 compared to other herpesviruses because the open reading frame (ORF) products and mechanisms of HSV-1 capsid formation were studied early on. Although several HSV-1 capsid maturation-related ORF homologs have been identified in other herpesviruses, many aspects of their molecular functions remain unclear.

Four types of capsids are formed during HSV-1 capsid maturation; these four forms have been defined as the procapsid, A-capsid, B-capsid, and C-capsid (Fig. 2). In the first step of capsid formation, the major capsid protein and scaffold protein (also called assembly protein) form a heterodimer, and these heterodimers self-assemble to form the procapsid. The procapsid, which has a spherical outer layer consisting of major capsid proteins and a spherical inner layer consisting of scaffold proteins, is a porous and hollow particle.^1,2) In other words, the outer layer of the procapsid is held by an inner scaffold layer, and the outer layer and inner layer interact together through a non-covalent bonding interaction.³⁾ Next, when the protein-protein interaction between the outer layer and the inner scaffold layer is detached by cleavage of the scaffold mediated by the viral protease, the spherical scaffold layer remains inside the outer layer, and the outer layer undergoes spontaneous angularization, becoming icosahedral. This icosahedral capsid with a spherical scaffold layer is referred to as the B-capsid.^4–6) Both procapsids and B-capsids retain the inner spherical scaffold layer, and it is difficult to distinguish between procapsids and B-capsids in images obtained by general transmission electron microscopy (TEM). The A-capsid is an empty icosahedral capsid that does not contain the scaffold protein.^7,8) When the scaffold proteins are successfully ejected from either the procapsids or B-capsids, and the single-unit genome processed by the terminase complex is properly packaged, pro- or B-capsids can be converted into a mature capsid, which is referred to as the C-capsid.^7,8)

Fig. 2. HSV-1 Capsid Morphologies

Procapsid: the first type of capsid formed during the capsid maturation process. The procapsid is a spherical and porous capsid. It contains the scaffold inner layer composed of scaffold proteins which attach to the outer layer composed of major capsid proteins. B-capsid: an icosahedral capsid that does not contain the viral genome. The B-capsid contains a spherical scaffold layer inside that is detached from the outer layer of the capsid. A-capsid: an icosahedral capsid, lacking the scaffold proteins and lacking the viral genome. C-capsid: an icosahedral capsid lacking the scaffold proteins but containing the single-unit-length viral genome. The C-capsid is the normal mature capsid.

The terminase complex components of each HHV are shown in Table 1. The terminase complex is comprised of three different viral proteins. In HSV-1, pUL15, pUL28, and pUL33 form a terminase complex, and other HHVs also form terminase complexes comprised of their respective homologous proteins. In HSV-1, six terminase complexes form a ring, which functions by inserting viral DNA into the center of this hexameric ring.

Table 1. Terminase Complex Components of Each HHV

	Subfamily	Terminase complex components
HSV-1	α	pUL15	pUL28	pUL33
HSV-2	α	pUL15	pUL28	pUL33
VZV	α	pORF45/42	pORF30	pORF25
EBV	γ	BGRF1/BDRF1	BALF3	BFRF1A
HCMV	β	pUL89	pUL56	pUL51
HHV-6	β	U66	U40	U35
HHV-7	β	U66	U40	U35
KSHV	γ	ORF29	ORF7	ORF67.5

3. THE TERMINASE COMPLEX OF ALPHAHERPESVIRINAE (HSV-1, HSV-2, VZV)

The viral genome DNA homology of HSV-1 and HSV-2 is very high and most of the studies have focused on the HSV-1 terminase complex. The HSV-1 terminase complex is composed of pUL15, pUL28, and pUL33, which form a tripartite complex. Six tripartite complexes are thought to be assembled into a ring surrounding the viral DNA precursor.⁹⁾ HSV-1 pUL15 is not required for viral genome replication but is required for genome packaging into capsids. A UL15 temperature-sensitive (ts) mutant forms a large number of B-capsids containing scaffold proteins but no DNA at the non-permissive temperature (NPT) (i.e., in the pUL15-inactivated state).¹⁰⁾ The UL15 ts mutant is unable to cleave the viral genome at the NPT, and viral genome precursors are accumulated in infected cells.¹¹⁾ The UL15 gene is a spliced gene consisting of two exons separated by an intron.^12,13) Two UL15 gene products of 81 kDa and 30 kDa have been identified. The 81 kDa product (pUL15) is likely a component of the HSV-1 terminase complex, because the 81 kDa product is required for DNA cleavage and packaging.^14,15) pUL15 also has putative ATP-binding motifs referred to as the Walker A box and the Walker B box. The Walker A box is essential for pUL15 function.¹⁶⁾ In contrast to pUL28 and pUL33 described below, pUL15 has a nuclear localization signal (NLS) and can localize alone to the nucleus, the site of capsid maturation.¹⁷⁾ In addition, pUL15 has a nuclease domain required for viral genome cleavage and packaging, and pUL15 is likely the nuclease activity of the HSV-1 terminase complex.^18,19)

Similar to pUL15, HSV-1 pUL28 is not required for viral genome replication. A UL28 ts mutant fails to package and cleave the viral genome at the NPT. Moreover, the UL28 ts mutant fails to form mature capsids and instead forms B-capsids at the NPT.²⁰⁾ pUL28 interacts with pUL15.²¹⁾ Although pUL28 localizes to the cytoplasm, coexpression with pUL15 allows pUL28 to translocate to the nucleus from the cytoplasm.²¹⁾ Furthermore, pUL28 binds to single-stranded DNA containing the pac 1 sequence, which is present in the TRs of the viral DNA. The pac 1 sequence and the structure-specific DNA binding ability of pUL28 are important for cleavage and encapsidation of the viral genome.²²⁾ pUL28 also interacts with pUL33 as described below.²³⁾ Since pUL15 and pUL33 do not directly interact, pUL28 functions as a hub molecule and mediates the indirect interaction between pUL15 and pUL33.²⁴⁾ In addition, pUL28 contains a zinc-finger motif that is important for DNA cleaving and DNA packaging functions in the HSV-1 terminase complex.¹⁹⁾ It has been hypothesized that the sequence-specific DNA binding ability of pUL28 determines the DNA cleavage site of the pUL15 nuclease activity at the appropriate location.

The HSV-1 UL33 ts mutant, as well as the UL28 and UL15 ts mutants all fail to form mature capsids and instead form B-capsids at the NPT.^25,26) In addition, the UL33 ts mutant is unable to cleave and package the viral genome, and this mutant is unable to produce infectious virus. pUL33 interacts with pUL28 and enhances the interaction between pUL15 and pUL28.²⁴⁾ pUL33 is a low molecular weight protein with a total length of 130 amino acids, and its main function is unknown.

The tertiary structure of the HSV-1 terminase complex has been revealed by cryo-electron microscopy, cryo-EM.⁹⁾ One molecule each of pUL15, pUL28, and pUL33 form a tripartite complex, and six of these tripartite complexes assemble into a ring. Since there is a channel with an inner diameter of 39 Å in the center of this ring, and the diameter of type-B double-stranded DNA is 20 Å, it has been proposed that the viral genome moves within this central channel.

The VZV terminase complex consists of pORF45/42, pORF30, and pORF25. A region of exon II from ORF45/42 encodes the C-terminus of pORF45/42, which interacts with pORF30.²⁷⁾ pORF25 interacts with the region of pORF42 encoded by the second exon of ORF45/42 and pORF25 also interacts with pORF30.²⁸⁾ ORF25-deficient VZV is unable to produce infectious virus.²⁹⁾ Functional analysis of ORF30-deficient or ORF30-mutated VZVs revealed that pORF30 was required for viral genome cleavage and packaging, and the zinc-finger motif of pORF30 was important for viral production.³⁰⁾ The HSV-1 homolog of the VZV pORF30 is pUL28. Interestingly, in contrast to the B-capsids produced by the HSV-1 UL28 ts mutant at the NPT, ORF30-deficient and ORF30-mutanted VZVs formed capsids packed with crystal-like structures.³⁰⁾ To date, this form of capsid has not been characterized or defined.

4. THE TERMINASE COMPLEX OF BETAHERPESVIRINAE (HCMV, HHV-6A, HHV-6B, HHV-7)

The HCMV terminase complex consists of pUL89, pUL56, and pUL51. In HCMV lacking the UL89, UL56, or UL51 genes, progeny virus production is suppressed.^31,32) pUL56 translocates to the nucleus and is required for the correct nuclear localization of pUL51 and pUL89.^32,33) Both pUL56 and pUL89 have sequence-nonspecific nuclease activity in vitro, and the nuclease activity of pUL89 is Mn²⁺-dependent.^34,35) In addition, pUL89 does not have ATPase activity, but pUL56 has ATPase activity, and this ATPase activity of pUL56 is further enhanced by pUL89.³⁶⁾ pUL56 binds to the AT-rich DNA sequences, pac 1 and pac 2.³⁷⁾ When UL51 is knocked down in the HCMV genome, mature capsids are not formed, and instead B-capsids are formed.³⁸⁾ Furthermore, pUL51 contains a leucine-zipper domain that is important for viral proliferation.³⁹⁾ An interesting finding regarding the HCMV terminase complex was that letermovir (which has received manufacturing and marketing approval in Japan in 2018 as an anti-HCMV drug) was found to inhibit the HCMV terminase complex by genomic analysis of resistant viruses.⁴⁰⁾ Letermovir inhibits HCMV genome cleavage and increases B-capsid formation.⁴⁰⁾ It was reported that letermovir treatment exhibited high anti-HCMV activity and had a low frequency and severity of adverse events.⁴¹⁾

There have been no previous reports on the terminase complexes of HHV-6A, HHV-6B, and HHV-7, and their functions are still unknown.

5. THE TERMINASE COMPLEX OF GAMMAHERPESVIRINAE (EBV, KSHV)

The EBV terminase complex consists of BGRF1/BDRF1, BALF3, and BFRF1A. Deletion of BGRF1/BDRF1 and BFRF1A in the EBV genome inhibited EBV progeny virus production, and the capsid formation pathway was arrested at the B-capsid stage.⁴²⁾ Knockdown of BALF3 also suppressed progeny virus production, induced the accumulation of B-capsids, and reduced the amount of viral genome replication.⁴³⁾

The components of the KSHV terminase complex are ORF29, ORF7, and ORF67.5. ORF29 has Mn²⁺-dependent nuclease activity.⁴⁴⁾ ORF29 is involved in infectious virus production, viral genome replication, and expression of viral late genes.⁴⁵⁾ ORF7 interacts with ORF29 and ORF67.5, however ORF29 and ORF67.5 do not interact directly.⁴⁶⁾ ORF7 can translocate to the nucleus, whereas ORF29 alone and ORF67.5 alone lacks this ability. ORF7 is required for nuclear translocation of ORF29 and ORF67.5.⁴⁶⁾ KSHV deficient in ORF7 or ORF67.5 is unable to produce infectious virus and lacks the TR cleavage ability.^46–48) The interaction between ORF7 and ORF29 is enhanced by ORF67.5, and the interaction between ORF7 and ORF67.5 is also enhanced by ORF29.⁴⁸⁾ ORF7 harbors a zinc-finger motif, which is essential for KSHV virion production and for proper viral genome cleavage.⁴⁷⁾ Additionally, conserved regions of ORF67.5 in HHV homologs are important for the function of ORF67.5 in virus production.⁴⁸⁾ Interestingly, KSHV lacking ORF7 or ORF67.5 form a soccer ball-like capsid with a morphology different from the B-capsid.^47,48) This soccer ball-like capsid is formed after the procapsid in the KSHV capsid maturation process.⁴⁷⁾ This suggests that the soccer ball-like capsid is an immature capsid in which the collapsed scaffold protein products are not eliminated from the capsid and remain inside the capsid.

6. CONCLUSION

Many anti-herpesvirus drugs developed to date, such as acyclovir and ganciclovir, target viral DNA synthesis. However, these drugs have problems with side effects such as nephrotoxicity and myelosuppression, and there is also the possibility of the emergence of drug-resistant viruses. To address these issues, the development of anti-viral drugs with novel mechanisms of action is necessary. The mechanism of the herpesvirus terminase complex, which is responsible for packaging and cleavage of the single-unit-length viral genome into the capsid, may be an excellent target for the development of novel highly virus-specific anti-viral drugs because this mechanism is not present in humans. Letermovir, an HCMV terminase inhibitor already developed and in clinical use, has high anti-viral activity and few side effects. Unfortunately, letermovir has no inhibitory effects on HHV terminases other than HCMV.⁴⁹⁾ However, it is hoped that the elucidation of the function of each HHV terminase complex and the discovery of additional terminase inhibitors will lead to the development of new anti-herpesvirus drugs.

Acknowledgments

Y.I. was supported by the Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan and the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists. This work was partially supported by grants from the JSPS Grant-in-Aid for Scientific Research (18K06642 [M.F.] and JP22KJ2988 [Y.I.]).

Conflict of Interest

The authors declare no conflict of interest.

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