Human Chromosomal Integration of Latent State Human Herpes Virus 6 (HHV-6)

From MicrobeWiki, the student-edited microbiology resource

By: Kerri-Lynn Conrad

Introduction

Figure 1. Transmission electron micrograph visualization of Human Herpes Virus-6 (HHV-6) on the surface of a human lymphocyte. HHV-6 is the causative agent of roseola, a disease that effects nearly every human infant.


Genomic Structure of HHV-6

Figure 2. Genomic organization of HHV-6B. The asterisk indicates the start of lytic genomic replication. Viral telomeric sequences are indicated by green bars T1 and T2. Open arrows represent protein coding regions.


HHV-6 is an enveloped, linear double stranded DNA virus. The genome is 160 to 162 kB in size, with a 143-144 kB unique region (1, 2). The unique region is flanked by 8-9 kB terminal direct repeats at each end, which contain repeats of the hexanucleotide sequence GGGTTA, identical to the telomeric sequences of vertebrate chromosomes (3, 4).

The genome has a cluster of seven gene blocks that are conserved among all herpesviruses, as well as the US22 gene family, conserved in other β-herpesviruses such as CMV and HHV-7 (5, 6). Although the function of the US22 gene family is not fully understood, it has been found that members of this gene family may act as transactivators, enhancing and increasing HHV-6 viral gene expression (7).

The products of HHV-6 genes U27, U41, U43, U74, U77, and U73 are involved in viral replication. The protein product of gene U27 encodes the DNA polymerase processivity factor, binding to DNA polymerase and thus, enhances the rate of viral genome transcription. Gene U41 encodes a single stranded DNA binding protein, also facilitating DNA polymerase's interaction with the viral genome. The protein products of genes U43, U74, and U77 produce a viral helicase-primase complex that facilitates replication of the viral genome. Additionally, the origin-binding protein produced by gene U73 also aids in genomic DNA replication. HHV-6 attachment to host cells is facilitated by viral surface proteins gB and gH, encoded by viral genes U39 and U48, respectively (8).

Unique to HHV-6 among all other herpesviruses are genes U83 and U94. Gene U83 encodes a chemokine that uses signaling mechanisms such as calcium fluxes to recruit lymphocytes and macrophages for productive or latent infection (9). Gene U94 encodes protein product RepH6, which binds the human TATA-binding protein, where it has an effect on regulation of viral gene expression and viral DNA replication. Furthermore, RepH6 has been shown to play a role in latent stage infections, as it is transcribed in latently infected lymphocytes (10).

There are two variants of HHV-6; HHV-6A and HHV-6B. HHV-6A and HHV6B share an overall nucleotide sequence identity of approximately 90%. HHV-6A has 110 ORFs, while HHV-6B has 119 ORFs. Major genomic differences between the two strains lie in the direct repeat regions near the ends of the viral genome, as well as in the proteins encoded by genes U86-U100 in the early stage of infection. The differences observed in early stage viral proteins are the result of the splicing patterns unique to each viral variant (11, 12).

HHV-6 Replication Cycle

Figure 3. Replication cycle of HHV-6.

To initiate viral replication, HHV-6A and HHV-6B glycoproteins gH, gL, gQ, and gB attach to host cell surface receptor CD46. All nucleated cells in the human body contain CD46, and thus, the range of host cells for HHV-6 is expansive. The nucleocapsid of HHV-6 is then transported to the host cell's nucleus via the cellular microtubule network. Viral DNA is then released into the nucleus (12).

Expression of the viral genome requires utilization of host transcriptional and translational machinery. Like other herpesviruses, both variants of HHV-6 express initial early (IE), early (E), and late (L) stage proteins. IE proteins, encoded by viral genes U86, U89, U95, U16-U-19, and U3, are expressed immediately after infection and are involved in regulation of subsequent viral gene expression. Early proteins, encoded by viral genes such as U27, U41, U43/U74/U77, U73, and U94 are required for replication of the viral genome and virion assembly. Late stage proteins often become incorporated into mature virions. An example of a late HHV-6 protein is that encoded by viral gene U83, which helps to establish latent infection (8, 9, 12).

Viral DNA replication takes places in the host cell nucleus, by means of the rolling circle mechanism characteristic of herpesviruses. Virions are packaged and assembled within the host nucleus. Virus particles bud out of the nucleus into the cytoplasm, where acquire tegument proteins encoded by viral genes U31 and U54. After tegument acquisition, the virions travel to the Golgi, where they are enveloped with glycoproteins gH, gL, gQ, and gB. Mature HHV-6 particles are then released from the cell via exocytosis (Figure 3).

Epidemiology

Seroprevalence of HHV-6 is estimated to range between 90-95% in the adult population of the world. It has been shown that antibody titers and thus seropositivity can decrease with increasing age (13). Seroprevalence in the world is rapidly approaching 100%, with the exception of Morocco, where only 20% of individuals are seropositive (14).

Initial HHV-6 infection usually occurs within the first 6 to 15 months of life, with peak infection rates observed in infants 6 to 9 months of age, after the protection of maternal antibodies cease (15). Although there is no serologic test to distinguish HHV-6A and HHV-6B, it is believed that HHV-6B is more seroprevalent, causing the majority of clinically observed infections. HHV-6A seropositivity is usually observed in immunocompromised individuals or in adult patients who display clinical signs of exanthema subitum (roseola) (8, 12, 16).

HHV-6 transmission likely occurs through saliva, as HHV-6 can replicate readily in the epithelial cells of the salivary glands. This mode of transmission is especially likely for HHV-6B, as almost all saliva samples analyzed for HHV-6 contain the HHV-6B variant (5, 17). Germ line transmission of HHV-6 can occur, and is observed in approximately 2% of births (18).

Pathophysiology of HHV-6 Infection

Figure 4. HHV-6 infection results in roseola, often diagnosed by a characteristic red rash. The roseola rash can form as either raised bumps or can be flat. It is non-contagious and is usually found on the neck, abdomen, back, and trunk, but can also be present on the arms and legs

There are 3 recognized stages of HHV-6 infection. Initial HHV-6 infection most often occurs within children 6 to 15 months old and is generally the only symptomatic period most infected individuals will experience. Latent infection persists in the lymphocytes and monocytes of healthy adults and children. The virus shows no sign of pathology during this stage and individuals remain asymptomatic. Although infrequent, reactivation of the latently infected virus or re-infection in immunocompromised individuals can occur, causing a recurrence of symptoms [8, 12].

Initial Infection

HHV-6 infection observed in infants and young children is almost always caused by variant HHV-6B. HHV-6B can infect a wide range of host cells because of the prevalence of host cell receptor CD46. However, HHV-6B most often replicates in CD46 coated CD4+ T lymphocytes during initial infection(8).

Initial infection is characterized by a febrile illness, with fever temperatures nearing or exceeding 40°C (104°F). HHV-6 infection accounts for 10-40% of hospital admissions of infants with high fevers (19). The fever can last for 3-7 days and then rapidly terminates. After cessation of the fever, approximately 17% of infected infants develop exanthema subitum, commonly known as roseola (20). The rash persists for 1-2 days, and is not a means of transmission. The rash is most often confined to the back, trunk, and neck, but can appear on the face, arms, and legs, generally causing no discomfort to the child (21).

In addition to high fever and in fewer cases, the development of exanthema subitum, HHV-6 infection can cause fatigue, gastrointestinal tract and respiratory tract symptoms. Approximately 10% of children in the initial stage of HHV-6 infection experience febrile seizures (12). High levels of viral titer have been isolated from the brain and cerebrospinal fluid, and can be associated with rare CNS symptoms, such as encephalitis (8, 12).

Figure 5. Stages of HHV-6 Infection

Latency in Healthy Children and Adults

HHV-6 latency and persistence in healthy, seropositive adults and children characterizes the second stage of HHV-6 infection. Viral replication continues in the epithelial cells of the salivary glands during this stage. This accounts for the abundance of HHV-6 isolates in saliva samples, and also represents the primary means of transmission to seronegative individuals (22).

Latency is achieved in host lymphocytes, monocytes, and possibly some organ tissues. HHV-6 DNA can be detected in the peripheral blood mononuclear cells (PBMCs) of 90% of seropositive individuals. HHV-6 achieves latency through integration into specific sites of the host chromosome (23).

Reactivation in Immunocompromised Individuals

The most acute clinical manifestations of HHV-6 infection are observed during reactivation of the virus in immunocompromised individuals. Such immunosuppression is often observed in individuals receiving organ transplants, as immunocompetency is reduced therapeutically to decrease chances of transplant rejection (12). Reactivation is also observed clinically in AIDS patients. HHV-6 has been shown to increase HIV replication by up-regulating certain cytokines and transactivating the long terminal repeats of the HIV genome, leading to increased viral loads and more aggressive AIDS onset (24).

Latency via Human Chromosomal Integration

Figure 6.FISH visualization of HHV-6 integration into the telomere region of chromosome 9 in metaphase and interphase cells.

HHV-6 is unique among human herpesviruses in that it is able to persist in latent state by naturally integrating into regions of the host's chromosomes. HHV-6 chromosomal integration is observed in approximately 2% of seropositive individuals, and is often the cause of high levels of viral DNA in host serum and plasma. HHV-6A and HHV-6B have both been found to integrate into the human genome, yet independently; co-integration has never been observed. Integration of HHV-6A is observed in nearly a third of congenital infections, although the rate of initial infection in the general population is 2-3%. Therefore, it is suggested that the primary route of HHV-6A transmission is through the germ line (25, 26). In cases of HHV-6 chromosomal integration, viral DNA can be isolated from every cell in the body, including skin fibroblasts, hair follicles, and blood cells (25). Immunocompetent patients with viral chromosome integration are identified by comparing the HHV-6 viral load in the host's blood and serum. Although viral loads will be substantial in both samples, concentration in serum will be at least 50-fold lower than the blood isolates (27).

HHV-6 has already been shown to integrate into 7 different chromosomal sites. In every case analyzed, chromosomal integration is always found in the telomere regions of the host chromosome, on either the p or q arm of one homolog of the chromosome, although not confined to a particular chromosome (25). Human telomeres contain repeats of the sequence TTAGGG. These repeats function to protect the ends of the chromosomes from degradation due to exonucleases. Additionally, these repeat sequences prevent shortening of the chromosome during replication. The genome of HHV-6 contains complementary GGGTTA repeats in the direct repeat regions. The repeats in the HHV-6 genome are only 21 to 80 base pairs in length, and are thus significantly shorter than the repeat sequences observed in human telomeres (28). The mechanism of HHV-6 integration is likely due to homologous recombination between the telomere repeat sequences of the human chromosome and the complementary repeat sequences of the HHV-6 genome. It has been previously shown that the telomere regions of the human chromosome are unstable and prone to such homologous recombination events (25). Another proposed mechanism of HHV-6 chromosomal integration involves the RepH6 viral protein encoded by gene U94. RepH6 can bind DNA site-specifically, and has also been shown to function as an endonuclease, helicase, and ATPase. Such functions of RepH6 mirror the chromosomal integration techniques of the related adeno-associated virus (AAV) rep gene. Therefore, RepH6 may facilitate chromosomal integration in a manner similar to the rep gene of AAV (25)

Several specific sites of HHV-6 chromosomal integration have been identified using FISH, chromosome specific PCR sequencing, and Gardella gel techniques (25, 26, 27, 29). A study of 9 British individuals, 7 adults, and 2 children, showed ubiquitous HHV-6 chromosomal integration. Only one site of HHV-6 integration was observed in each individual. This particular study identified chromosome sites 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4 has sites of HHV-6 integration (25). A more recent study identified telomeric sites 17p13.3, 18q23, and 22q13.3 as sites of HHV-6 integration. In this study, sites of HHV-6A integration were conserved among family members, suggesting possibility of vertical transmission (29). Both studies were able to induce reactivation and latent viral replication via treatment with established viral reactivating compounds.

HHV-6 chromosomal integration has been observed in malignant lymphomas. It is suggested that integrated HHV-6 DNA could be oncogenic, modifying the expression of host cell proteins involved in cell growth and apoptotic signaling (30).

Although HHV-6 is the only human herpesvirus known to integrate into host chromosomes, Marek's disease virus, an avian herpesvirus, also integrates into host chromosomes. Marek's virus also integrates into host chromosomes via similar complement telomeric sequences. Phylogenetic analysis of HHV-6 and Marek's disease genomes show no close genetic relationship. However, the similar complement telomeric repeats GGGTTA are shared by both viruses, reinforcing these sites as the mechanism of chromosomal integration (25, 31).

Vertical Transmission of Integrated HHV-6 through Host Germ Line

Figure 7.FISH on metaphase chromosomes using a HHV-6 specific probe. Panel A represents a lymphoma patient, panel B is her husband, and panel C are the chromosomes of their daughter. The arrow and arrowhead in panel C indicate that the daughter inherited integrated HHV-6 from each of her parents at distinct loci: 1q44 from her father, and 22q13 from her mother.

Although the primary route of HHV-6 transmission is thought to be from mother to child through saliva, several case studies have demonstrated that vertical transmission to offspring through the germ line is also possible. In a study of four families with high HHV-6 viral loads, FISH and chromosome specific PCR sequencing at viral-chromosomal junctions has shown that HHV-6A can be transmitted through the germ line (29). HHV-6B was also shown to be vertically transmitted in case studies of four families. Several individuals in these families suffered from similar autoimmune disorders and even lymphomas, suggesting clinical inheritance (32). FISH and hair follicle analyses of congenitally infected infants and their parents have shown that chromosomal integration of HHV-6 causes approximately 86% of such congenital infections. Statistically, this suggests that 1 out of every 116 live-born infants exhibits chromosomally integrated HHV-6 (26). It has also been shown that the offspring of individuals with chromosomally integrated HHV-6 can inherit the virus at two different loci, one from each parent, once more reinforcing the mechanism of vertical transmission (Figure 7, 33).

Future Work

References

Edited by Kerri-Lynn Conrad, student of Joan Slonczewski for BIOL 375 Virology, 2010, Kenyon College.