Latent infection and reactivation of human cytomegalovirus

Mini Review

Latent i n f e c t i o n and r e a c t i v a t i o n of human cytomegalovirus K Numazaki, H Asanuma, S Chiba Department of Pediatrics, School of Medicine, Sapporo Medical University, Sapporo, Japan

Summary Human cytomegalovirus (HCMV) is the most common cause of congenital and perinatal infections throughout the world. Primary infection with HCMV usually follows a benign course, but the virus remains latent or persistent in the host cell thereafter. Under immunosuppressive conditions, latent or persistent infection can be reactivated to produce a wide variety of clinical manifestations. Understanding the epidemiology of HCMV infection is a key element in development of strategies for prevention of infection. Definition of sites and mechanisms involved in the maintenance of latent or persistent HCMV infection and reactivation is also essential for a thorough understanding of the pathogenesis of HCMV infection. This mini review focuses on recent advances in the study of persistent infection and reactivation of HCMV. Although the actual sites of latency or persistence of HCMV infections are still controversial, peripheral blood mononuclear cells (PBMC) and endothelial cells appear to be the principal site of infection. Persistent infections caused by HCMV could be augmented by a decrease in major histocompatibility complex (MHC) expression as well as by virus-mediated immune dysfunction. It is also likely that specific cellular interactions as well as production of several cytokines are necessary for the reactivation of HCMV. Key words: Human cytomegalovirus, latent infection, reactivation, major histocompatibility complex, macrophages, monocytes Serodiagn. Immunother. Infect. Disease 1995, Vol. 7, 70-74, June

Introduction

H u m a n cytomegalovirus (HCMV) genome is doublestranded DNA, 230 kilobases in size, the largest human beta-herpes virus and has unique short and long regions. H C M V has the largest genome encoding more than 200 potentially immunogenic proteins. H C M V is an antigenically and structurally diverse group of viruses which are closely related to herpes simplex, varicella zoster, and Epstein-Barr (EB) viruses. The characteristic of latency and reactivation is shared with other members of the herpesvirus family. After infection, the viral genome is transcribed in a regulated sequence, resulting in the serial transcription of three different classes of mRNA, immediate early (IE), early (E) and late (L). IE (transactivator) is among the Received: 8 November 1994 Accepted: 18 November 1994 Correspondence and reprint requests to: K Numazaki, Department of

Pediatrics, School of Medicine, Sapporo Medical University, S.1 W.16 Chuo-ku, Sapporo, 060 Japan 0888-0786/1995 Elsevier Science B.V.

initial viral proteins expressed and is present in abundance at 2-6 h of infection. IE gene expression is necessary for the expression of other H C M V gene expression and initiating the viral cycle in latently infected cells. E (non structural) proteins expressed from 6-24 h post infection and finally L (structural) proteins produced after viral D N A replication. HCMV is the most common cause of congenital and perinatal infections throughout the world. However, the prevalence of congenital HCMV infection ranges widely among different populations. Almost 90% of the population in Japan is seropositive by 20 yr of age. As a result of transmission during the course of delivery, by ingestion of infected breast milk and by blood transfusion, perinatal infections are much more prevalent than congenital infections. Perinatal H C M V infection often involves the hepatobiliary tract but rarely causes clinical manifestations in normal individuals. Generally, HCMV infections are effectively controlled by the immune system without the ultimate clearance of the virus. Seropositivity for antibodies against H C M V is indicative of latent infection, but insufficient as a predictor for the

Numazaki et al.: Latency and reactivation of HCMV

risk of recurrence. T cell immunity is of central importance in recovery from primary H C M V infection and protection against reactivation. In immunocompromised hosts, however, H C M V involvement of the liver and pancreas may be extensive, causing significant functional impairment. On the basis of the results of clinical and serological studies of H C M V excretors, it was postulated that many of the infected infants experience hepatitis, hepatosplenomegaly or a mononucleosis syndrome. As clinical diagnosis of H C M V infection is generally not easy, rapid and accurate laboratory diagnosis of H C M V infection is required for appropriate patient management. Transfusions containing leucocytes have been found to be a significant source of H C M V infection in bone marrow and graft recipients. The incidence of transfusion-acquired H C M V infection can be remarkably reduced by using leucocyte-depleted blood products. It was also reported that filtration was successful in removing H C M V D N A with analysis of pre- and postfiltration samples by polymerase chain reaction (PCR)L Definition of sites and mechanisms involved in the maintenance of latent H C M V infection and reactivation is essential for a thorough understanding of the pathogenesis of H C M V infection. Establishment of latent infection

Among herpes viruses, both H C M V and EBV may shed in oropharyngeal secretions for prolonged periods during convalescence from illness or as a manifestation of subclinical infection. There is a risk of recurrence of neonatal H C M V infection, whereas the risk is low when primary infection occurred at adult age 2. Latent H C M V burden and risk of recurrence were reported to be related to the extent of virus multiplication during primary infection. H C M V latency differs from herpes simplex virus latency by its wide organ distribution. The presence of latent H C M V in multiple organs provides the molecular basis for recurrence from multiple organs. After primary infection, H C M V becomes latent, and peripheral blood mononuclear cells (PBMC) appear to be one of the principal sites of persistent infection3.4. Monocytes are one site of carriage of H C M V genome in healthy carriers. Although extended culture of monocytes with or without specific differentiation signals can result in substantial numbers of cells permissive of H C M V infection, freshly isolated monocytes are difficult to infect with H C M V and do not replicate the virus. Peripheral blood of healthy carriers usually does not contain infectious HCMV. Freshly isolated monocytes from healthy seropositive H C M V carriers do not produce IE, E, or L H C M V R N A but the differentiation of these cells to monocyte-derived macrophages results in induction of IE gene expression 5. Some degree of differentiation may be necessary for permissive infection of freshly isolated human monocytes with H C M V 6,7.

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During active infection with HCMV, viral antigen is consistently present in peripheral blood leucocytes 8. Detection of H C M V in the polymorphonuclear leucocyte fraction of peripheral blood has been reported in patients infected with human immunodeficiency virus (HIV) 1 and in other immunocompromised patients 2. Although several reports on PCR testing for H C M V in healthy blood donors have been published, it was unclear which types of cells can harbour the virus and support productive infection and whether peripheral blood lymphocytes behave as specific carriers of the virus during systemic infection or represent a reservoir of replicating virus or a possible site of latency 4. Peripheral blood monocytes are difficult to infect with H C M V in vitro, and viral gene expression cannot be reproduced in the peripheral blood cells of healthy carriers as mentioned above 9,1°. During an acute infection with CMV, both mononuclear and polynuclear cells have been found to express H C M V antigens 11. However, it is hard to detect viral antigens in the lymphocytes of immunocompromised hosts, who usually have a few peripheral blood mononuclear cells and characterization of H C M V antigen-positive T lymphocytes was possible in non-immunocompromised infants with liver dysfunction associated with perinatal primary H C M V infectionlL Activation of CD8+ and CD4+ lymphocytes requires recognition of viral antigens as short peptides bound to major histocompatibility complex (MHC) class I and class II antigens on the surface of antigen-presenting cells (APC), such as monocytes and macrophages. H C M V can both increase and decrease expression of M H C class I and II antigens. First, it was reported that H C M V bound 13:-microglobulin and it was reported that M H C class I molecule was the receptor for H C M V 13. Subsequent studies suggested that there was no correlation between M H C class I expression and susceptibility to H C M V infection. H C M V U L 18 gene product which could bind to 132-microglobulin is not necessary for HCMV-induced defective M H C class I assembly. Interference with normal M H C class I assembly and expression may have implications for restriction of the diversity of the CD8+ cytotoxic T lymphocytes (CTL) repertoire directed against H C M V antigens 14. The presence of HCMV-infected endothelial cells in the peripheral blood of patients with an active H C M V infection indicates the association of widespread occult vascular damage 15. HCMV-related transplantation atherosclerosis has been recognized as a major cause of allograft failure in long-term cardiac transplant recipients. H C M V is thought to induce an inflammatory reaction of polymorphonuclear leucocytes against arterial endothelial cells. Endothelial cells are one of the major targets for H C M V infection and may also represent a site of persistence. Recently, Sedmak et al. 16reported that H C M V can downregulate the expression of M H C class II glycoproteins induced by interferon--/ (IFN-~) on infected endothelial cells. HCMV-infected APC, such as monocytesmacrophages or endothelial cells, would be unable to

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present viral antigens acquired exogenously and endogenously. Similar alterations in MHC class I glycoprotein expression have been reported 17. These facts provide potential mechanisms for the persistence of HCMV infection by evasion of host immunosurveillance. Persistent infections caused by HCMV could be augmented by a decrease in MHC expression as well as by virus-mediated immune dysfunction. HCMV reactivation and the roles of cells of the immune system

HCMV infection is reported to cause transient immunosuppression. Such immunological perturbation may assist this agent in establishing persistent infections with severe clinical outcomes. The impaired responses of T lymphocytes associated with HCMV infection were corrected with exogenous interleukin-2 (IL-2). These impaired responses were reported to be restricted to the T cell receptor/CD3 activation pathwayTM. HCMV infection is a major complication after organ transplantation and in immunosuppressed patients 8. Those most at risk are seropositive prior to transplantation and typically reactivate latent virus 4-8 weeks after transplantation. During HCMV infection, alloreactive CTL were found in high frequencies and they appeared concomitantly with the virus-specific CTL. It has been proposed that upregulation of MHC antigen expression by HCMV infection may result in activation of alloreactive CTL. A high incidence and mortality of HCMV pneumonia has been reported in bone marrow transplant patients who fail to develop HCMV-specific pre-CTL responses. Following primary infection, production of IgG and IgM antibodies, responses by CTL, activation of natural killer (NK) cells, and antibody-dependent killer cells (ADKC) occur. CTL specific for HCMV can be isolated from PBMC of healthy HCMV antibody-positive individuals. Alteration of MHC class I expression could affect lysis of HCMV-infected endothelial cells by CD8+ CTL. A decrease in MHC expression may not only enhance persistence of HCMV infection but establishment of reactivation. IE, glycoprotein B (gB) and nonenvelope structural virion such as the matrix protein pp65 have been shown to serve as target antigens for the MHC class I-restricted CDS+ CTL ag. Responses of CTL and NK cells to HCMV also represent the predominant mechanism necessary for resistance to and recovery from HCMV infection2°. It was reported that HCMV evolved a unique mechanism for selectively limiting the presentation of the potentially immunogenic IE protein, which might preclude IEspecific CTL from providing protective immunity to HCMV infection21. An alternative basis for the alloreactivity and autoimmunity induced by MHC class II antigen expression could be molecular mimicry of HLA-DR by the IE-2 antigen of HCMV. The IE-2 protein has immunological crossreactivity with HLADR, in that antibodies generated against this IE-2 polypeptide react with the HLA-DR 13 chain.

Activation of CD8+ cells was associated with recovery from both primary and secondary infections and with a low risk of relapse after antiviral therapy. Increased numbers of subsets of CD8+, CD57+ cells correlated with previous HCMV infection22. HCMV infection after transfusion may be caused by exogenous donor virus and not the reactivation of endogenous virus in the recipients. However, blood transfusion itself may also reactivate HCMV. Transfusion of uninfected allogeneic blood is associated with reactivated HCMV in latently-infected mice and humans, which suggests that transfusion alone is sufficient stimulus for HCMV reactivation. Immunological reaction to foreign antigen appears to be important in latent HCMV reaction, as exemplified by the reactivating effects of histoincompatible cocultivation and allogeneic blood transfusion. HCMV hepatitis is often recognized in both normal and immunocompromised hosts and in patients with both primary and reactivated HCMV infections. Although infantile HCMV hepatitis was speculated to be caused by primary infection in the perinatal period, immunological conditions of the hosts may modify the clinical manifestations. We investigated the role of peripheral blood mononuclear cells, especially CD8+ T lymphocytes, in infants with liver dysfunction associated with perinatal primary HCMV infection, by flow cytometry and PCR 12. Expression of HCMV antigens in CD8+ cells was also found in patients with liver dysfunction associated with perinatal primary HCMV infection. HCMV infection of CD8+ cells may play an important role in the pathogenesis of CD8-activation. Extrahepatic HCMV infection producing an immunological reaction may also be necessary to give rise to neonatal and infantile hepatitis. It is likely that specific cellular interactions as well as other cytokines are necessary for HCMV reactivation. HCMV infection induces polyclonal CTL activation, not only against the challenge virus but also against alloreactive CTL and memory CTL from a previous exposure to other viruses. This was thought to be mediated by cytokines as a consequence of HCMV infection. Once activated, CD4+ T lymphocytes functioned through cytokines such as IFN-~ and interleukin-10 (IL-10) in regulation of virus-specific antibody production by B lymphocytes and in modulation of CD8+ T lymphocytes. Cytokines can override the suppressive effects of viruses on MHC antigen expression. Interleukin-6 (IL-6) regulates immune responses, haematopoiesis and acute phase reactions. It has been shown that several viruses are capable of activating the IL-6 promoter in a variety of cell types. It was also reported that HCMV can cause a perturbation of cytokine cascades involved in the regulation of IL-6 by endothelial cells23. HCMV can also significantly reduce the augmenting effects of IFN-',/ on MHC class II glycoprotein and mRNA expression. Reactivation of HCMV could also occur by stimulation of the HCMV IE promoter. Tumour necrosis factor-or (TNF-c,) stimulated the IE enhancer/promoter activity

Numazaki et aL: Latency and reactivation of HCMV

in a transfected h u m a n monocytic cell line. Since premonocytic cells are suggested to be the site of H C M V latency, T N F - a may also have a potential pathophysiological significance in H C M V reactivation 24. It was also reported that the H C M V I E genes upregulated T N F - a gene activity 25. Activation of T N F gene expression by the H C M V I E gene products m a y account for the inflammatory response associated with H C M V infections.

Conclusions H C M V is an agent which causes serious disease in infants who have acquired the virus in utero, and in patients who are i m m u n o s u p p r e s s e d due to HIV-1 infection, organ transplantation and i m m u n o s u p p r e s sive chemotherapy. Understanding the epidemiology of H C M V infection is required for the d e v e l o p m e n t of strategies for prevention and therapy. T h e actual sites of latency or persistence of H C M V infections and the factors controlling latency and reactivation are still controversial. H C M V can both increase and decrease expression of M H C class I and II antigens. Persistent infections caused by H C M V could be a u g m e n t e d by a decrease in M H C expression as well as by virusmediated i m m u n e dysfunction. It is likely that specific cellular interactions as well as other cytokines are necessary for H C M V reactivation. Further studies, including clarification of the mechanisms of persistence and reactivation or kinetic studies of H C M V infections, are necessary.

Acknowledgements This work was supported by research grants from the Ministry of Education, Science and Culture of Japan and the Clinical Pathology Research Foundation of Japan.

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