Epigenetic regulation of the life cycles of oncogenic viruses

by Signe MacLennan

There are some viruses that drive cancer development and progression. These oncogenic viruses can promote genomic instability and inflammation and have also been linked to epigenetic changes in the viral and host genomes, thereby contributing to malignancy. Here we explore how these viruses use epigenetics as part of their normal life cycles, how these epigenetic processes are disrupted in cancer, and how we may be able to therapeutically target these epigenetic changes in the future.

Some viruses can drive cancer

Viruses are infectious agents that rely upon their hosts to replicate and ultimately infect new hosts¹. Whether or not viruses are “alive” is a matter of debate, but regardless, viruses may inflict serious harm to their hosts. The genetic material (or genomes) carried by viruses encode instructions for making more viruses (i.e. virus progeny). Depending on the virus, the viral genome can be DNA or RNA and may exist in double- or single-stranded forms. Viruses can also have circular or linear genomes depending on the virus type¹.

A small subset of viruses are able to infect humans and a subset of these have oncogenic (i.e. cancer promoting) potential² ^,3. There are seven viruses capable of driving cancer in humans currently recognized by the World Health Organization (WHO). These oncogenic viruses include human papillomavirus (HPV), Epstein-barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), Kaposi sarcoma herpesvirus (KSHV), human T-lymphotropic virus 1 (HTLV-1), and Merkel cell polyomavirus (MCPyV)^2,3. In addition, sometimes human immunodeficiency virus (HIV) is included as the eighth oncogenic virus⁴. Some oncogenic viruses only drive one particular cancer type, as is the case of MCPyV, which drives Merkel cell carcinoma, but others, like EBV, are more generalist and have been linked to multiple cancer types³.

Oncogenic viruses drive cancer in a multitude of ways, including by inducing inflammation, deregulating the immune system, and inducing mutations in the host (i.e. human) genome³. More recently, we have learned ways by which these viruses have adopted epigenetic mechanisms to progress through their life cycles and how these processes are disrupted in cancer². Importantly, cancer is a rare side effect of infection with these viruses, as often cancer limits the ability of the virus to infect new hosts¹.

Viral life cycles

Clearly, viruses are highly diverse and as such, they have highly diverse life cycles. However, viruses often share life cycle features, including one or multiple latent phases and a lytic phase³. Notably, the lytic and latent phases are shared among many viruses, but some viruses, like EBV, have highly complex life cycles, with multiple distinct latent phases⁵.

The lytic phase describes the phase in which a virus uses the host’s cellular machinery to make new virus progeny in order to infect new cells and new hosts. The lytic cycle culminates in the destruction of the host cell by breaking the new virus progeny through the host cell membrane. The destruction of the host’s cell membrane (i.e. the barrier between the cell and its environment) is also called lysis, hence the name⁵.

Latency or the latent cycle, describes a separate phase of the virus life cycle, wherein the virus “waits” within host cells for a trigger to initiate the lytic cycle. In the latent phase, a virus often incorporates parts of its genomes into that of the host in a process known as viral integration⁶. For example, in the case of HIV infection, viral integration is the major barrier preventing cure for patients living with HIV, as a reservoir of integrated viruses persist in host cells and the viral genome is replicated alongside the host genome each time the infected host cell divides⁶. In this way, the virus remains hidden within host cells and is invisible to the host’s immune system⁷.

Epigenetics as part of viral life cycles

Epigenetics translates to “on top of genetics” and describes the ways in which DNA (or RNA) and chromatin are modified without directly changing the genetic sequence⁸. Epigenetic modifications often influence how the genome is organized and regulated; without such regulation the creation of different cell types would be impossible. Thus, all cells in the body have the same genome, but what differs is how this genome is read out and interpreted (via the epigenome), which ultimately leads to different gene expression patterns between different cell types⁸.

In much the same way that our cells use epigenetic modifications to determine cell fate, viruses use epigenetics to progress through their life cycles¹ ^,2. In fact, although viruses are considered relatively simple and primitive agents, they have been found ways to use DNA methylation, recruit host histone modifying enzymes, and encode for microRNAs as part of their life cycles. Here we highlight a few examples of the epigenetic modifications and enzymes that add epigenetic marks that are utilized by viruses as part of viral life cycles¹ ^,2.

DNA and RNA methylation in viral life cycles

A methyl group can act as an epigenetic mark when added to DNA or RNA and can influence which genes are turned on (i.e. are expressed) and off (i.e. are repressed)⁸. Depending on the proximity to a gene, DNA methylation can have opposing effects on gene expression. For instance, when DNA methylation occurs in an upstream control region called the promoter region, it is often repressive, whereas when DNA methylation occurs within the gene body itself, it correlates with the gene being expressed (transcribed⁸).

Among oncogenic viruses, DNA and RNA methylation can influence whether the virus stays in the latent phase or progresses to the lytic phase. For instance, in the case of EBV infection, EBV virions are often highly methylated in their latent phases, but become unmethylated in the lytic phase, so lytic genes can be expressed⁹.

Methylation can also influence whether the host cell transforms into a cancer cell¹⁰. In the case of HPV-driven malignancies, three HPV genes are especially important for progression to cancer¹⁰. The viral oncogenes, E6 and E7, encode viral oncoproteins that degrade host cellular proteins that otherwise inhibit the formation of cancer (i.e. the tumour suppressor proteins p53¹¹ and Rb¹²). When these important host cellular proteins can no longer function properly, the host cell can gain cancerous characteristics including resisting cell death and dividing uncontrollably. However, as part of the normal HPV life cycle, E6 and E7 expression is regulated by another HPV gene called E2 and is actually normally kept at bay as E2 inhibits E6 and E7 expression⁹. Thus, when E2 function is blocked, E6 and E7expression is left unchecked, thereby leading to malignancy. One of the ways E2 function can be blocked is via DNA methylation. Specifically, to inhibit E6 and E7 expression, E2 normally binds to E2 binding sites on the HPV genome, but when these sites become aberrantly methylated, E6 and E7 expression increases. In this case, DNA methylation serves as a physical barrier preventing E2 function (Figure)⁹.

Example of how viral (HPV) methylation can be disrupted to promote cancer development — An example of how viral methylation can be disrupted to promote cancer development. Here the example is the methylation of E2 binding sites in HPV driven cancer development (e.g. cervical cancer). E2 binding sites are usually unmethylated in the HPV genome, but can become methylated as part of cancer development. When the E2 binding sites are unmethylated, E2, a negative regulator of the viral oncogenes, E6 and E7, can bind to the E2 binding site and suppress E6 and E7 expression. However, when the E2 binding site is aberrantly methylated, E6 and E7 can be expressed as E2 can no longer bind to the E2 binding sites to suppress the expression of E6 and E7. Note that there are other HPV genes in the HPV genome, but they are not pictured here.

Other epigenetic modifications to DNA and RNA as part of viral life cycles

Besides DNA methylation, we are just learning about other ways DNA and RNA can be decorated with different epigenetic marks. In the case of HBV, N6-methyladenosine (m6A) is an epigenetic mark that can be added to viral RNA¹³. There is now evidence that both the m6A mark and its location are important factors influencing how HBV progresses through its life cycle and potentially influencing the transformation of the host cell into a cancer cell¹³.

Histone modifications in viral life cycles

In human cells, there is over two metres of DNA, but all that DNA has to fit within the confines of a microscopic cell nucleus¹⁴. In order to achieve the level of compaction required to fit all the DNA into such a small volume, DNA is wrapped around proteins called histones. Depending on whether the DNA is wrapped tightly or loosely around histones, influences which genes are expressed. Histone marks such as histone acetylation and histone methylation can affect the tightness of DNA wrapping¹⁴. For instance, histone acetylation promotes increased gene expression, as it neutralizes a positive charge and since DNA is negatively charged, this loosens the otherwise tight association between the histone and the DNA¹⁴.

Different enzymes within human cells are responsible for adding, taking away, and interpreting histone marks and these enzymes can be hijacked by viruses for both host and viral gene expression modulation¹⁵ ^,16. For instance, an EBV protein called EBNA2 recruits enzymes that acetylate histones (called histone acetyltransferases - HATs) to viral and host promoters to promote the expression of certain genes¹⁵. LANA, an KSHV protein, interacts with histone enzymes that remove acetyl groups (called histone deacetylases - HDACs), to promote gene repression and to either maintain latency or enter the lytic phase, depending on the specific enzyme type^16,17.

Viral microRNAs

Another component of the epigenome concerns that of non-coding RNAs (ncRNAs). As their name suggests, ncRNAs are RNAs that do not code for proteins and instead are themselves functional molecules⁸. One particular class of ncRNAs is microRNAs (miRNAs), which bind to specific parts of coding RNAs (i.e. messenger RNA - mRNA) to inhibit their translation into proteins¹⁸. Both EBV and KSHV express high levels of miRNAs as part of viral latency¹⁹ ^,20. In the case of EBV, viral miRNAs serve many functions in maintaining latency, including repressing viral genes and thus preventing EBV from being recognized by the immune system, as well as repressing host genes that are cancer promoting, and ultimately contributing to the latent to lytic phase switch¹⁹ ^,20.

Viral integration in cancer and how it may influence the epigenome

Some oncogenic viruses incorporate parts of their genomes into the host genome as part of cancer development, in a process called “integration”^21,22. It is not entirely understood why some regions of the host genome are more likely to have integration happen there, but sometimes viruses integrate near cancer-promoting genes or in regions of the genome more unstable and thus susceptible to breakage²¹ ^,22. In HPV infection, viral integration can disrupt the E2 gene, thereby promoting high E6 and E7 oncogene expression, but there is also emerging evidence that viral integration may influence the surrounding epigenome²³. For instance, different classes of HPV integration were found to affect the surrounding DNA methylation patterns in distinct ways²³.

How may viral epigenetic reprogramming be targeted in the treatment of cancer?

Some of the current research on oncogenic viruses concerns how we can use what we know about how oncogenic viruses use epigenetics to better target virally driven malignancies. It has been proposed, and there is evidence to support the use of HDAC inhibitors for the treatment of EBV+ cancers²⁴. The rationale here is that inhibiting HDACs will cause EBV to enter the lytic phase and kill the cancer cell it is inhabiting. Another future potential treatment for some virally driven malignancies is DNMT1 inhibitors²⁵. The DNMT1 gene codes for a protein that maintains methyl groups to DNA as it replicates. In using DNMT1 inhibitors for the treatment of some virally driven cancers, the ways oncogenic viruses hijack DNMT1 expression may be blocked, and the cancer cell may be better recognized by the immune system. However, although promising, current epigenetic cancer drugs are not without side effects, as they are targeting host proteins that are essential for cellular functions²⁶.

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