Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Open reading frame 45 (ORF45) of Kaposi''s sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.Kaposi''s sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus and the causative agent of several human cancers, including Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease (3, 6). Like all herpesviruses, KSHV has two alternative life cycles, a latent and a lytic cycle. During latency, only a few viral genes are expressed, and no progeny viruses are produced. Under appropriate conditions, latent viral genomes are activated, initiate lytic replication, and express a full panel of viral genes, in a process that leads to viral assembly, release of progeny virus particles, and de novo infection of naïve cells (3, 6). KSHV establishes latent infection in the majority of infected cells in cases of KS, primary effusion lymphoma, and multicentric Castleman''s disease, but lytic replications occur in a small fraction. The recurrent and periodic lytic cycles of KSHV are believed to play critical roles in viral pathogenesis (6, 7).Open reading frame 45 (ORF45) is a KSHV-encoded gene product that plays a critical role in the viral lytic cycle. It is an immediate-early protein and is also present in viral particles as tegument protein (26, 27, 30). Disruption of ORF45 has no significant effect on overall viral lytic gene expression or DNA replication in BAC36-reconstituted 293T cells induced with both tetradecanoyl phorbol acetate (TPA) and sodium butyrate together, but the ORF45-null mutant produces 5- to 10-fold fewer progeny viruses than the wild type and the mutant virus has dramatically reduced infectivity, suggesting that ORF45 plays important roles at both early and late stages of viral infection (29). In addition to its roles as a tegument component, which are possibly involved in viral ingress and egress processes, KSHV ORF45 interacts with cellular proteins and modulates the cellular environment. At least two such functions have been described. First, KSHV ORF45 inhibits activation of interferon regulatory factor 7 (IRF-7) and therefore antagonizes the host innate antiviral response (28). Second, KSHV ORF45 interacts with p90 ribosomal kinase 1 and 2 (RSK1/RSK2) and modulates the extracellular signal-regulated kinase/RSK signaling pathway, which is known to play essential roles in KSHV reactivation and lytic replication (12). All of these data suggest that KSHV ORF45 is a multifunctional protein.ORF45 is unique to the gammaherpesviruses; it has no homologue in the alpha- or betaherpesviruses. ORF45 homologues have been identified as virion protein components in other gammaherpesviruses, such as Epstein-Barr virus (EBV), rhesus rhadinovirus (RRV), and murine herpesvirus 68 (MHV-68), suggesting that certain tegument functions of ORF45 are conserved (2, 11, 18). ORF45 homologues differ in protein length. KSHV ORF45 is the longest, at 407 amino acids (aa); RRV, EBV, MHV-68, and herpesvirus saimiri (HVS) have proteins of 353, 217, 206, and 257 aa, respectively. The limited homologies lie mostly at the amino- and carboxyl-terminal ends. The middle portion of KSHV ORF45 diverges from those of its homologues. The homologues differ in subcellular localization. We and others have reported previously that KSHV ORF45 is found predominantly in the cytoplasm (1, 21, 28, 30), whereas ORF45 of MHV-68 is found exclusively in the nucleus (9). Recently, we found KSHV ORF45 also present in the nuclei of BCBL-1 cells in what resembled viral replication compartments, suggesting that ORF45 could shuttle into the nucleus (12).Nucleocytoplasmic trafficking of proteins across the nuclear membrane occurs through nuclear pore complexes. Small molecules of up to approximately 9 nm in diameter, corresponding to a globular protein of approximately 40 to 60 kDa, can in principle enter or leave the nucleus by diffusion through nuclear pores (15, 17, 24). Large molecules are transported with the aid of a related family of transport factors, importins and exportins, which recognize nuclear localization sequence (NLS)-containing or nuclear export sequence (NES)-containing proteins (15, 17, 23). CRM1 (exportin 1) has been identified as a common export receptor that recognizes human immunodeficiency virus Rev-like leucine-rich NES sequences and is responsible for the export of such NES-containing proteins (4, 5, 19, 22). CRM1-dependent nuclear export is specifically inhibited by a pharmacological compound, leptomycin B (LMB), that interacts with CRM1 and thus blocks such NES-mediated protein export (4).To understand the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we attempted to locate the signals that control its subcellular localization. In the research reported here, we identified a leucine-rich NES and an adjacent basic NLS in KSHV ORF45. We demonstrated that the regulated intracellular trafficking of ORF45, especially its translocation into the nucleus, is important for KSHV lytic replication.     

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II^−/− (CII^−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII^−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII^−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII^−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII^−/−, CD40^−/−, or CD80/86^−/− mice, compared with that in wild-type or CD28/CTLA4^−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II^−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II^−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2D^b-restricted), and ORF 61 (p79, H-2K^b-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II^−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L^−/− mice (7) and CD40^−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II^−/− mice to MHV-68-infected class II^−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.     

Time-Dependent Transformation of the Herpesvirus Tegument

All herpesviruses have a layer of protein called the tegument that lies between the virion membrane and the capsid. The tegument consists of multiple, virus-encoded protein species that together can account for nearly half the total virus protein. To clarify the structure of the tegument and its attachment to the capsid, we used electron microscopy and protein analysis to examine the tegument of herpes simplex virus type 1 (HSV-1). Electron microscopic examination of intact virions revealed that whereas the tegument was asymmetrically distributed around the capsid in extracellular virions, it was symmetrically arranged in cell-associated virus. Examination of virions after treatment with nonionic detergent demonstrated that: (i) in extracellular virus the tegument was resistant to removal with Triton X-100 (TX-100), whereas it was lost nearly completely when cell-associated virus was treated in the same way; (ii) the tegument in TX-100-treated extracellular virions was asymmetrically distributed around the capsid as it is in unextracted virus; and (iii) in some images, tegument was seen to be linked to the capsid by short, regularly spaced connectors. Further analysis was carried out with extracellular virus harvested from cells at different times after infection. It was observed that while the amount of tegument present in virions was not affected by time of harvest, the amount remaining after TX-100 treatment increased markedly as the time of harvest was increased from 24 h to 64 h postinfection. The results support the view that HSV-1 virions undergo a time-dependent change in which the tegument is transformed from a state in which it is symmetrically organized around the capsid and extractable with TX-100 to a state where it is asymmetrically arranged and resistant to extraction.All herpesviruses have a tegument, a layer of protein located between the virus membrane and the capsid. Depending on the virus species, the tegument can be 20 to 40 nm in thickness, and it may be uniformly or asymmetrically distributed about the capsid (7, 17, 24, 33). The tegument is composed predominantly of virus-encoded proteins that together can account for up to half or more of the total virion protein mass. Tegument proteins are thought to be those involved in the early stages of infection before progeny virus proteins are synthesized.The tegument has been most thoroughly studied in herpes simplex virus type 1 (HSV-1). Examination of virions by electron microscopy has demonstrated that the tegument is not highly structured. Its morphology is described as predominantly granular with fibrous elements also present (7, 19). Analysis by cryo-electron microscopy, followed by icosahedral reconstruction has shown that the tegument is not icosahedrally ordered, although a small amount of tegument density is observed close to the capsid surface at the pentons (3, 47).The HSV-1 tegument is composed of approximately 20 distinct, virus-encoded protein species whose amounts vary considerably. The predominant components are UL47, UL48, and UL49, each of which occurs in more than 800 copies per virion (8, 46). In contrast, others, such as RL2 (ICP0), RS1 (ICP4), UL36, and UL37, occur in ∼100 copies or less. Trace amounts of host cell-encoded proteins are also present (15). Many of the tegument proteins are required for virus replication (34), and functions have been defined for most (9, 12, 31, 40).Biochemical studies have demonstrated that the tegument makes noncovalent contacts with both the virus capsid and the membrane. Studies of capsid-tegument contacts have emphasized binding of UL36, a tegument protein, to UL25, a capsid protein located near the vertices and involved in DNA encapsidation (5, 20, 29). Other tegument proteins such as UL48 (VP16), UL37, and UL49 (VP22) are found to associate with UL36 and may be bound to the capsid indirectly by way of UL36 (13, 44). UL16 binds reversibly to the capsid while UL46 (VP11/12) has been shown to bind to both the membrane and the capsid (21, 22, 26). Binding of tegument proteins to the membrane has been shown to occur by way of attachment to UL11 (45) and also to the internal domains of membrane glycoproteins, including glycoprotein D (gD), gH, and gE (4, 6, 11).We describe here the results of a study in which electron microscopy and protein analysis were used to clarify the structure of the HSV-1 tegument and its attachment to the capsid. The study was designed to extend the observation that most of the HSV-1 tegument remains attached to the capsid when the membrane is removed from the virus by treatment with nonionic detergent (19). Cell-associated and extracellular virions were compared after treatment with Triton X-100 (TX-100).     

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.Human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]), the prototypical betaherpesvirus, is ubiquitous in humans and establishes a persistent infection in the host (19). HCMV also reinfects healthy seropositive individuals, suggesting another mechanism for maintaining persistence in a population (9). Intrauterine transmission and HCMV infection of the developing fetus constitute a leading viral cause of birth defects (32). HCMV is also a leading cause of opportunistic infections in immunocompromised patients, including transplant recipients and patients with AIDS (10, 20). HCMV infection has also been implicated as a cofactor in such diverse diseases as atherosclerosis and cancer (8, 17, 33, 66).HCMV replicates its genome in the nucleus, and acquisition of the final tegument and envelope is thought to occur in the cytoplasm of infected cells (73, 77). Envelopment of HCMV has been reported to occur by budding into cytoplasmic vacuoles that are composed of HCMV glycoproteins required for the assembly of infectious virions (37). The fully mature virus is released from the cell through either exocytosis or, possibly, lysis of the infected cells (56). The nucleic acid-containing capsid is embedded in a proteinaceous tegument layer that occupies the space between the nucleocapsid and the envelope. The tegument contains approximately 40% of the virion protein mass and approximately 20 to 25 known virion proteins, most of which are phosphorylated (40, 44). The assembly pathway and protein interactions required for formation of the tegument layer and the role of individual tegument proteins in the replication and assembly of infectious HCMV remain poorly understood. Deletion of viral genes encoding some tegument proteins results in varying levels of impairment in virus production (11-13, 35, 43, 45, 53, 68). Some tegument proteins, such as pp28 (pUL99) and ppUL25, are expressed only in the cytoplasm of infected cells during HCMV replication, whereas others, such as ppUL53 and pp65 (pUL83), are expressed in the nuclei of cells early in infection but are localized predominantly in the cytoplasm late in infection (68). Others, such as the tegument protein ppUL69, are expressed only in the nuclei of infected cells. Finally, the intracellular localization of other tegument proteins, such as pp150 (pUL32), is less well defined in that both nuclear and cytoplasmic localizations have been described (34, 68).HCMV pp150 (basic phosphoprotein [BPP], pUL32) is the 1,048-amino-acid product of the UL32 gene of HCMV and an abundant constituent of the HCMV virion. Homologues of pp150 are found in other betaherpesviruses, including chimpanzee CMV, rat CMV, mouse CMV, HHV-6, and HHV-7, but not in alpha- or gammaherpesviruses (2). It is expressed late in HCMV infection (15, 68). It comprises 9.1% of infectious virion mass and 2% of the mass of dense bodies, suggesting that it is preferentially incorporated into virions (87). It has an estimated molecular mass of 113 kDa and is posttranslationally modified by phosphorylation and glycosylation, resulting in a molecular mass of 150 kDa in purified virus preparations analyzed by SDS-PAGE (41, 42, 65). pp150 has been classified as a tegument protein based on its presence in virion preparation, noninfectious enveloped particles, and cytoplasmic nucleocapsids but not in immature nuclear capsids (27, 28, 40). It has been suggested that pp150 contacts the capsids through the distal end of the capsomeres or through the triplex subunits that interlink them (16, 86). It has been reported to bind HCMV capsids in vitro through its amino one-third (6). We have also noted association of pp150 with the virion capsid by cryo-immunoelectron microscopy (W. Britt and H. Zhou, UCLA, Los Angeles, CA, unpublished findings). In primary human foreskin fibroblast (HFF) cells infected with HCMV, pp150 accumulates in a juxtanuclear structure that is termed the assembly compartment (AC), which colocalizes with markers of the distal secretory pathway and with other tegument proteins, including pp28 and pp65 and envelope glycoproteins gB, gH, and gM/gN (68). The virus-induced AC appears to overlap with microtubules emanating from the microtubule-organizing center (MTOC) and is proposed to be a cytoplasmic site of virion assembly (37, 68).The function of pp150 is unknown, although its close association with the nucleocapsid suggests potential involvement in nuclear targeting during entry and in nuclear targeting of the encapsidated viral DNA, capsid tegumentation, and/or envelopment late in infection. It is essential for production of infectious virus, since the deletion of the UL32 open reading frame (ORF) leads to loss of virus replication and has been reported to be important in cytoplasmic maturation of HCMV, especially in viral egress (2, 22, 84, 91, 92). In cells infected with ΔUL32 virus, which lacks pp150, fewer virus particles accumulated in the cytoplasm, although nuclear steps in virus assembly were not affected (84). It was also observed that in the absence of pp150, nucleocapsids were present in the viral assembly compartment but failed to proceed further to vesicle transport-associated release (84). These observations, together with pp150 abundance in the virion, suggest a primary contribution for this structural protein in the morphogenesis and/or cytoplasmic transport of progeny virion particles to sites of virion envelopment.Since pp150 has no predicted intracellular trafficking signals, its localization to the AC in virus-infected cells has been postulated to be dependent on interactions with cellular and/or viral proteins. Using yeast two-hybrid (Y2H) screening experiments we identified the cellular protein Bicaudal D1 (BicD1) as an interacting cellular protein. Bicaudal D was originally defined as a Drosophila protein that is involved in establishing the asymmetric cytoplasm in the developing oocyte (82, 89). Two homologues of Bicaudal D, BicD1 and BicD2, have been reported in humans, and these proteins have been reported to be involved in dynein-mediated microtubule transport as well as in COPI-independent Golgi-endoplasmic reticulum (ER) transport (38, 39, 55). Microtubule-dependent transport is an energy-dependent active transport system that includes both positive-end (directed away from the MTOC) and negative-end (directed toward the MTOC) transport. The direction of transport depends on cargo interactions with the molecular motors directing this transport, with dynein being associated with negative-end transport and kinesin with positive-end transport. BicD1 colocalizes with Rab6a in the trans-Golgi network and on cytoplasmic vesicles that associate with Golgi membranes in a Rab6-dependent manner secondary to a Rab6 binding domain at the C terminus of BicD1, suggesting an important role for BicD1 as an adaptor for dynein-dependent transport in the cell (55). In addition to having a role in the Golgi-ER trafficking, BicD1 has been shown to regulate anchoring of microtubules to the centrosome, as BICD1/2 knockdown induced microtubule unfocusing, with microtubules no longer appearing to radiate from the centrosome (26). BicD1 binds to its cargo via its C-terminal domain and to the dynein motor via its N-terminal domain (38). In this study we demonstrated that pp150 and BicD1 interact and that this interaction was required for localization of pp150 to the AC in virus-infected cells. In addition, we demonstrated that inhibition of BicD1 expression by short hairpin RNA (shRNA) led to a reduction in the yield of infectious virus. Finally, we demonstrated that formation of the AC and the assembly of infectious virions were dynein dependent, suggesting a critical role in microtubules in the production of infectious HCMV. Together, these results argue that HCMV replication is dependent on efficient localization of pp150 to the AC through its interaction with BicD1 and that pp150 localization to the AC is dynein dependent.     

Protective and Pathologic Roles of the Immune Response to Mouse Hepatitis Virus Type 1: Implications for Severe Acute Respiratory Syndrome

Intranasal mouse hepatitis virus type 1 (MHV-1) infection of mice induces lung pathology similar to that observed in severe acute respiratory syndrome (SARS) patients. However, the severity of MHV-1-induced pulmonary disease varies among mouse strains, and it has been suggested that differences in the host immune response might account for this variation. It has also been suggested that immunopathology may represent an important clinical feature of SARS. Little is known about the host immune response to MHV-1 and how it might contribute to some of the pathological changes detected in infected mice. In this study we show that an intact type I interferon system and the adaptive immune responses are required for controlling MHV-1 replication and preventing morbidity and mortality in resistant C57BL/6J mice after infection. The NK cell response also helps minimize the severity of illness following MHV-1 infection of C57BL/6J mice. In A/J and C3H/HeJ mice, which are highly susceptible to MHV-1-induced disease, we demonstrate that both CD4 and CD8 T cells contribute to morbidity during primary infection, and memory responses can enhance morbidity and mortality during subsequent reexposure to MHV-1. However, morbidity in A/J and C3H/HeJ mice can be minimized by treating them with immune serum prior to MHV-1 infection. Overall, our findings highlight the role of the host immune response in contributing to the pathogenesis of coronavirus-induced respiratory disease.Severe acute respiratory syndrome (SARS) is caused by a zoonotic coronaviral infection that reached epidemic proportions beginning in late 2002 (37, 52, 55, 76, 84, 86). The etiologic agent, SARS-coronavirus (CoV), is a novel group 2 CoV that emerged in the human population exposed to infected animals that were present in wet markets in various provinces of southern China (16, 22, 35, 45, 57, 61). Although the outbreak was quickly contained by the application of aggressive public health measures, it highlighted the deadly potential of this novel pathogen as more than 8,000 people in more than 25 countries were affected, and almost 800 infected individuals died (37, 76, 84, 86). Although there have not been additional outbreaks of this disease in the general population since 2003, due to the continued presence of related viruses in bats and other animals and to cultural practices prevalent in the local population in southern China, the reemergence of this pathogen in the human population may occur in the future (40).Currently, there are no rigorously tested efficacious prophylactic or therapeutic agents targeting this pathogen. Given the lethal potential of this virus, it is imperative to develop specific antiviral therapies that can be rapidly and universally applied. One of the serious drawbacks in the field is the paucity of appropriate animal models that faithfully reproduce the clinical features of SARS (52, 60). Although a mouse-adapted strain of this virus is available, studies with this strain need to be performed in biosafety level 3 facilities (48, 59). Logistical issues associated with such requirements hamper the rapidity and ease with which one can perform a comprehensive and detailed systemic examination of the dynamics of host-pathogen interactions. Recently, it was reported that intranasal infection of certain strains of mice with a related group 2 respiratory CoV, mouse hepatitis virus type 1 (MHV-1), induced pulmonary disease that was very similar to that observed in human subjects infected with SARS-CoV (11). In addition to the phylogenetic proximity of MHV-1 and SARS-CoV, they also share similarities in genome organization and in mechanisms of replication (63, 68). Hence, it is likely that the pathophysiology observed in MHV-1-infected mice mimics important pathological features associated with SARS-CoV infection in humans. A dysregulated immune response characterized by aberrant cytokine production is postulated to contribute to clinical disease in patients with SARS (8, 26, 55, 58, 72, 75, 82, 83). MHV-1 infection of susceptible strains of mice is also associated with an altered cytokine profile, and published reports suggest that the host immune response to the virus is an important contributor to the pathology observed in susceptible strains of mice (11). Examination of the immune response to a pathogen is critical for the purpose of designing rational and effective vaccination approaches. In addition, it also helps identify potentially deleterious effects of the immune response that can subsequently be manipulated to the advantage of the host, thereby maximizing recovery and minimizing morbidity.In the present study we have carried out a comprehensive analysis of the immune response to MHV-1 following intranasal infection of both resistant and susceptible strains of inbred mice. Our observations in alpha/beta interferon (type I IFN) receptor-knockout (IFN-αβR-KO) mice and NK cell-depleted mice shed light on the protective role of these components of the innate immune response in resistant C57BL/6J (B6) mice. And our examination of the adaptive immune responses to MHV-1 shows that they function as a double-edged sword, mediating protection in resistant strains and contributing to pathology in susceptible strains of mice.     

Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.The herpesvirus virion is composed of an icosahedral nucleocapsid containing the viral genome, an envelope of cellular origin with inserted viral (glyco)proteins, and a tegument which links nucleocapsid and envelope comparable to the matrix of RNA viruses. The herpesvirus tegument contains a multitude of viral and cellular proteins (reviewed in references 45 and 46). Tegument proteins execute various regulatory and structural functions, including activation of viral gene expression (2), modulation of the host cell for virus replication (26, 51, 55), and mediation of posttranslational modification of proteins (10, 27, 50). Numerous interactions have been identified among tegument proteins, between tegument and capsid proteins, and between tegument and envelope proteins (7, 14, 16, 18, 33, 36, 42, 53, 58-61).The largest tegument proteins found in the herpesviruses are homologs of pUL36 of herpes simplex virus type 1 (HSV-1). Pseudorabies virus (PrV) pUL36 consists of 3,084 amino acids (aa) with a molecular mass of 324 kDa (33). PrV and HSV-1 pUL36 are essential for viral replication (13, 15). In their absence, nonenveloped nucleocapsids accumulate in the cytoplasm. Whereas in several studies nuclear stages like cleavage and packaging of the viral DNA as well as nuclear egress were not found affected (13, 15), another study indicated an effect of pUL36 deletion on PrV nuclear egress (41).pUL36 homologs complex with another tegument protein, pUL37, as has been shown for HSV-1 (59), PrV (15, 33), and human cytomegalovirus (3, 23), and the interacting region on pUL36 has been delineated for PrV (33) and identified at the amino acid level for HSV-1 (47). Deletion of the pUL37 interaction domain from PrV pUL36 impedes virion formation in the cytosol but does not block it completely, yielding a phenotype similar to that of a pUL37 deletion mutant (31). This indicates an important but nonessential role for pUL37 and the pUL37 interaction domain in pUL36 in virion formation (15). In contrast, absence of pUL37 completely blocks virion formation in HSV-1 (11, 38).pUL36 is stably attached to the nucleocapsid (39, 43, 56), remains associated with incoming particles during transport along microtubules to the nuclear pore (21, 40, 52), and is required for intracellular nucleocapsid transport during egress (41). In contrast, absence of pUL37 delays nuclear translocation of incoming PrV nucleocapsids but does not abolish it (35). HSV-1 pUL36 is involved not only in transport but also in docking of nucleocapsids to the nuclear pore (9), and proteolytic cleavage of pUL36 appears to be necessary for release of HSV-1 DNA into the nucleus (24).Immunoelectron microscopical studies of PrV-infected cells showed that pUL36 is added to nucleocapsids prior to the addition of pUL37 (33). Since neither pUL36 nor pUL37 was detected on primary enveloped PrV virions, it was concluded that acquisition of tegument takes place in the cytoplasm (20). However, conflicting data exist whether pUL36 is present in the nucleus, and whether it is already added onto the capsids in this cellular compartment. Indirect immunofluorescence, immunoelectron microscopy and mass spectrometry of intranuclear capsids yielded discrepant results. By immunofluorescence HSV-1 pUL36 was detected both in the cytoplasm and in the nucleus (1, 42, 48). However, whereas one study detected the protein on nuclear C-capsids by Western blotting (6), it was not found by cryo-electron microscopy and mass spectrometry (57). In contrast, the C terminus of PrV pUL36 was suggested to direct pUL36 to capsid assemblons in the nucleus (37) by binding to capsid-associated pUL25 (8), although pUL36 could not be detected in the nucleus during PrV infection (33). These differing results in HSV-1 and between HSV-1 and PrV might be due to the fact that pUL36 could be processed during the replication cycle and that the resulting subdomains may exhibit selective localization patterns (24, 28).Amino acid sequence analyses of HSV-1 and PrV pUL36 revealed several putative nuclear localization signals (NLS) (1, 4, 5, 49). HSV-1 pUL36 contains four of these NLS motifs (49). Functionality in nuclear localization of a reporter protein was shown for the NLS motif at aa 425 (1). This motif is highly conserved in herpesvirus pUL36 homologs pointing to an important function (1). Besides this conserved NLS (designated in this report as NLS1), two other NLS motifs are predicted in PrV pUL36. One is located adjacent to NLS1 (aa 288 to 296) at aa 315 to 321 (NLS2), and a third putative NLS motif is present in the C-terminal half of the protein (aa 1679 to 1682; NLS3) (4). Whereas this may be indicative for a role for pUL36 inside the nucleus, NLS motifs might also be involved in transport to the nucleus along microtubules (54) and docking at the nuclear pore complex (49).The discrepancy in pUL36 localization and the putative presence of pUL36 cleavage products with specialized functions and localization prompted us to generate monospecific antisera covering the major part of PrV pUL36 and to study localization of PrV pUL36 by immunofluorescence during viral replication and after transient transfection and by immunoelectron microscopy of infected cells.     

Structure and Capsid Association of the Herpesvirus Large Tegument Protein UL36

The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (∼50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.All herpesviruses have a tegument, a layer of protein located between the virus capsid and membrane. The tegument accounts for a substantial proportion of the overall virus structure. Its thickness (30 to 50 nm), for example, may be comparable to the capsid radius, and tegument proteins can account for 40% or more of the total virion protein. Herpesvirus tegument proteins are thought to function promptly after initiation of infection, before expression of virus genes can take place (11, 13, 14, 21, 33, 37).Electron microscopic analysis of virions has demonstrated that the tegument is not highly structured (9, 22). It does not have icosahedral symmetry like the capsid, and it may be uniformly or asymmetrically arranged around the capsid (26). Tegument structure is described as fibrous or granular, and its morphology is found to change as the virus matures. Studies with herpes simplex virus type 1 (HSV-1), for example, indicate that the tegument structure is altered in cell-associated compared to extracellular virus (26).The tegument has been most thoroughly studied in HSV-1, where biochemical analyses indicate that it is composed of approximately 20 distinct, virus-encoded protein species. The predominant components are the products of the genes UL47, UL48, and UL49, with each protein present in 800 or more copies per virion (12, 40). Other tegument proteins can occur in 100 or fewer copies, and trace amounts of cell-encoded proteins are also present (17). Tegument proteins are classified as inner or outer components based on their association with the capsid after it enters the host cell cytoplasm. The inner tegument proteins (UL36, UL37, and US3) are those that remain bound to the capsid after entry, while the others (the outer tegument proteins) become detached (7, 18).The HSV-1 UL36 protein has the potential to play a central role in organizing the overall structure of the tegument. With a length of 3,164 amino acids, UL36 could span the thickness of the tegument multiple times. One hundred to 150 UL36 molecules are present in the tegument (12), and they are bound to the capsid by way of an essential C-terminal domain (2, 16). UL36 is able to bind the major tegument components by way of documented direct (UL37 and UL48) and indirect (UL46, UL47, and UL49) contacts (6, 15, 24, 38).Here we describe the results of studies designed to test the idea that UL36 serves to organize the tegument structure. Beginning with infectious virus, a novel method has been used to isolate capsids that contain UL36 and UL37 but lack the virus membrane and are depleted of all other tegument proteins. These capsids (T36 capsids) were examined by electron microscopy to clarify the structure of UL36 and UL37 molecules and their location on the capsid surface.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.