Virus reactivation: a panoramic view in human infections

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is 'quiescent' (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus.     

CRISPR/Cas9, a powerful tool to target human herpesviruses

Over 90% of the adult population is infected with one or multiple herpesviruses. These viruses are characterized by their ability to establish latency, where the host is unable to clear the invader from infected cells resulting in a lifelong infection. Herpesviruses cause a wide variety of (recurrent) diseases such as cold sores, shingles, congenital defects and several malignancies. Although the productive phase of a herpesvirus infection can often be efficiently limited by nucleoside analogs, these drugs are ineffective during a latent herpesvirus infection and are therefore unable to clear herpesviruses from the human host. Advances in genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 facilitates virus research and may hold potential to treat or cure previously incurable herpesvirus infections by directly targeting these viruses within infected cells. Here, we review recent applications of the CRISPR/Cas9 system for herpesviral research and discuss the therapeutic potential of the system to treat, or even cure, productive and latent herpesviral infections.     

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.     

Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread

Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues.     

体外抑制EB病毒药物的应用研究

EB病毒容易感染人,与人类许多疾病高度相关,严重威胁人类健康。目前尚无有效的预防EB病毒感染的措施且针对EB病毒的药物比较局限,故而对EB病毒感染进行治疗显得十分重要。近年来研究发现,许多药物在体外可对EB病毒有很好的抑制效果。仅就体外抑制EB病毒的药物,如核苷类似药、硼替佐米、寡脱氧核苷酸、乳铁蛋白、砷剂、维生素甲类化合物、亚硒酸钠、某些中草药等的近期研究现状进行综述。     

Non-Human Primate Model of Kaposi's Sarcoma-Associated Herpesvirus Infection

Since Kaposi''s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi''s sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.     

Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.     

Host factors involved in resistance to retroviral infection

Viral replication requires the help of host cell factors, whose species specificity may affect viral tropism. On the other hand, there exist host factors that restrict viral replication. The anti-viral system mediated by some of these restriction factors, which is termed intrinsic immunity and is distinguished from conventional innate and adaptive immunity, has been described as playing an important role in making species-specific barriers against viral infection. Here, we describe the current progress in understanding of such restriction factors against retroviral replication, focusing on TRIM5alpha and APOBEC, whose anti-retroviral effects have recently been recognized. Additionally, we mention cyclophilin A, which is essential for HIV-1 replication in human cells and may affect viral tropism. Understanding of these host factors would contribute to identification of the determinants for viral tropism.     

Crystal structure of the herpes simplex virus 1 DNA polymerase

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.     

Host range of Kaposi's sarcoma-associated herpesvirus in cultured cells

Difficulties in efficiently propagating Kaposi's sarcoma-associated herpesvirus (KSHV) in culture have generated the impression that the virus displays a narrow host range. Here we show that, contrary to expectation, KSHV can establish latent infection in many adherent cell lines, including human and nonhuman cells of epithelial, endothelial, and mesenchymal origin. (Paradoxically, the only lines in which we have not observed successful latent infection are cultured lymphoma cell lines.) In most latently infected lines, spontaneous lytic replication is rare and (with only two exceptions) is not efficiently induced by phorbol ester treatment-a result that explains the failure of most earlier studies to observe efficient serial transfer of infection. However, ectopic expression of the KSHV lytic switch protein RTA from an adenoviral vector leads to the prompt induction of lytic replication in all latently infected lines, with the production of infectious KSHV virions. These results indicate (i) that the host cell receptor(s) and entry machinery for KSHV are widely distributed on cultured adherent cells, (ii) that latency is the default pathway of infection, and (iii) that blocks to lytic induction are frequent and largely reside at or upstream of the expression of KSHV RTA.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.     

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.     

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi’s sarcoma-associated herpesvirus latent replication

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/−) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.