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4.12.2: DNA Oncogenic Viruses - Biology

4.12.2: DNA Oncogenic Viruses - Biology


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An estimated 15 percent of all human cancers worldwide may be attributed to viruses.

Learning Objectives

  • Outline DNA oncogenic viruses

Key Points

  • Both DNA and RNA viruses have been shown to be capable of causing cancer in humans.
  • Epstein-Barr virus, human papilloma virus, hepatitis B virus, and human herpes virus-8 are the four DNA viruses that are capable of causing the development of human cancers.
  • The presence of viral gene products in tumor cells that require them to maintain their unchecked proliferation, can provide important targets for directed therapies that specifically can distinguish tumor cells from normal cells.

Key Terms

  • oncogenic: Tending to cause the formation of tumors.

There are two classes of cancer viruses: DNA and RNA viruses. Several viruses have been linked to certain types of cancer in humans. These viruses have varying ways of reproduction and represent several different virus families.

DNA Oncogenic Viruses include the following:

  • The Epstein-Barr virus has been linked to Burkitt’s lymphoma. This virus infects B cells of the immune system and epithelial cells.
  • The hepatitis B virus has been linked to liver cancer in people with chronic infections.
  • Human papilloma viruses have been linked to cervical cancer. They also cause warts and benign papillomas.
  • Human herpes virus-8 has been linked to the development of Kaposi sarcoma. Kaposi sarcoma causes patches of abnormal tissue to develop in various area of the body including under the skin, in the lining of the mouth, nose, and throat or in other organs.

DNA tumor viruses have two life-styles. In permissive cells, all parts of the viral genome are expressed. This leads to viral replication, cell lysis and cell death. In cells that are non-permissive for replication, viral DNA is usually, but not always, integrated into the cell chromosomes at random sites. Only part of the viral genome is expressed. These are the early control functions of the virus. Viral structural proteins are not made, and no progeny virus is released.

The first DNA tumor viruses to be discovered were rabbit fibroma virus and Shope papilloma virus, both discovered by Richard Shope in the 1930s. Papillomas are benign growths, such as warts, of epithelial cells. They were discovered by making a filtered extract of a tumor from a wild rabbit and injecting the filtrate into another rabbit in which a benign papilloma grew. However, when the filtrate was injected into a domestic rabbit, the result was a carcinoma, a malignant growth. A seminal observation was that it was no longer possible to isolate infectious virus from the malignant growth because the virus had become integrated into the chromosomes of the malignant cells.


Human oncogenic viruses: nature and discovery

Seven kinds of virus collectively comprise an important cause of cancer, particularly in less developed countries and for people with damaged immune systems. Discovered over the past 54 years, most of these viruses are common infections of humankind for which malignancy is a rare consequence. Various cofactors affect the complex interaction between virus and host and the likelihood of cancer emerging. Although individual human tumour viruses exert their malignant effects in different ways, there are common features that illuminate mechanisms of oncogenesis more generally, whether or not there is a viral aetiology.

This article is part of the themed issue ‘Human oncogenic viruses’.

1. Introduction

Cancers are not contagious in the sense that they are not transmitted from patients to close contacts. But global studies reveal that about one in six cancers worldwide have an infectious aetiology [1,2]. Although this estimated attributable fraction is significant, it is probably a substantial underestimate since developing countries are particularly hard hit by viral cancers yet tend to have poor or nonexistent cancer registries. Some sites in Africa with high quality reporting, such as Kampala in Uganda [3], reveal that up to 50% of incident cancers are caused by infectious agents and that these cancers afflict a younger population than traditionally seen in North American or European settings. Some cancer registries exclude non-melanoma skin cancers, or attribute deaths to HIV rather than HIV-related cancers [4]. These estimates also tend to exclude new infectious causes for cancer (e.g. Merkel cell polyomavirus) or a new association of a known virus with a tumour for instance, Epstein–Barr virus (EBV) causes more cases of gastric carcinoma than lymphoma. Various infectious agents, such as bacteria (Helicobacter pylori in stomach cancer and mucosal-associated lymphomas) and helminths (carcinomas of the urinary bladder and gall bladder) are associated with malignancy, but approximately 1.6 million of the 2 million new cancer cases each year due to infection arise as a consequence of persistent infection by oncogenic viruses. This theme issue focuses on new developments for those viruses and their mechanisms of action.

The notion that a transmissible agent might play a role in some types of human cancer dates back to epidemiological observations made by the physician Domenico Rigoni-Stern in 1842 [5]. Analysing death certificates for women of Verona in 1760–1839, he noted that while nuns had an increased risk of breast cancer, they had a lower risk of cervical cancer compared to married women and much lower risk than sex workers. He concluded that the development of cervical cancer is related to sexual contact. Of course, there was no knowledge of hormones or viruses in Rigoni-Stern's time, but it seems a neat symmetry that 120 years later, the 1966 Nobel Prize for Physiology and Medicine was awarded on the one hand to Charles Huggins ‘For his discoveries concerning hormonal treatment of cancer’ and on the other to Peyton Rous ‘For his discovery of tumour-inducing viruses’. Isolation of Rous's eponymous sarcoma virus in chickens had been made 55 years earlier, representing the longest ‘incubation period’ between discovery and recognition in the annals of the Nobel Prizes [6]. An additional 42 years passed before Harald zur Hausen was similarly recognized for the discovery of strains of human papillomavirus (HPV) that cause cervical cancer [7]. Tumour viruses led the way in early molecular cell biology with Nobel awards to Renato Dulbecco, Howard M Temin and David Baltimore in 1975 for in vitro cell transformation by viruses and for reverse transcriptase to Baruch S Blumberg in 1976 for the elucidation of hepatitis B virus to J Michael Bishop and Harold E Varmus in 1989 for the cellular origin of retroviral oncogenes and to Richard Roberts and Philip Sharp in 1993 for the discovery of RNA splicing in adenovirus.

Tumour-inducing viruses have been of immense importance to our understanding of molecular carcinogenesis leading to the discovery of oncogenes and tumour suppressor genes. Oncogenes were first discovered in association with retroviruses and later applied to most forms of cancer [8–10]. Studies on human and animal cancer viruses led to discoveries that include over 30 cellular oncogenes, genes that are prefixed with a ‘c’ (e.g. c-myc, c-erb, c-fos), whereas oncogenes carried by viruses are prefixed with a ‘v’ (e.g. v-myc and v-ras). H-ras and K-ras were first found as viral oncogenes stolen from the host genome by the Harvey rat sarcoma and Kirsten rat sarcoma viruses respectively whereas N-ras was discovered directly in human tumours by DNA transfection. Before mutation analysis became standard, tumour suppressors and oncoproteins such p53 [11,12] and PI3 K [13] were identified by their association with tumour virus proteins. It is not an exaggeration to say that tumour virology formed the bedrock for all areas of modern cancer molecular biology.

Just over half a century has passed since first discovery of a human virus causing cancer, Epstein–Barr virus (EBV or HHV4) [14]. Now there are seven established human cancer viruses (table 1), with additional suspects being scrutinized. In this theme issue, the authors survey cancer viruses and give a broad understanding of the common features and the differences between the viruses. Lunn et al. [15] provide an overview on the epidemiology of viral tumours, outlining their common characteristics as well as their differences. Technologies have made enormous progress since 1964 and the complex interplay between viruses and their hosts is being uncovered at the molecular level: viral genomics by Tang & Larsson [16] innate immune evasion by Hopcraft & Damania [17] and viral regulation of DNA replication and repair by Pancholi et al. [18]. The recognition that viruses can cause cancer has led to two anti-cancer vaccines targeting infection by high-risk strains of human papillomaviruses (HPV) and by hepatitis B virus (HBV), as described by Stanley [19], which have already begun to reduce the incidence of these cancers in humans [20,21].

Table 1. Viruses involved in human cancer. ca., cancer ds., disease.

We have also been fortunate to recruit leading experts for each of the major cancer viruses as authors for reviews. The role of EBV in B-cell tumours is discussed by Shannon-Lowe et al. [22] and in undifferentiated nasopharyngeal carcinoma by Tsao et al. [23]. The related gamma-herpesvirus, Kaposi sarcoma herpesvirus (KSHV or HHV8), rose to prominence by causing an epidemic of cancer among AIDS patients and remains a scourge in Africa, parts of Asia and parts of South America as described by Mariggiò et al. [24]. We turn to the small human DNA viruses with a review by McBride [25] on HPV, emphasizing the novelties of their replication. DeCaprio [26] describes current understanding on the most recently recognized human tumour virus, Merkel cell polyomavirus (MCV), and other newly discovered human polyomaviruses that have been discovered by sequencing technologies. Liver cancer resulting from HBV and hepatitis C viruses (HCV) infection is an unsolved public health problem despite the success of the HBV vaccine and anti-viral drugs targeting HCV. Ringelhan et al. [27] describe these liver pathogens and point towards how the fundamental molecular virology of these viruses can provide new avenues for prevention and therapy. Finally, the human retroviruses, viruses with both RNA- and DNA-based life cycles, remain a fundamental source of new biology. Bangham & Matsuoka [28] describe human T lymphotropic virus type I (HTLV-I) while Kassiotis & Stoye [29] discuss endogenous human retroviruses. HIV contributes to each of these viral cancers by targeting the immune system and, because of this, it is considered a human carcinogen by the International Agency for Cancer Research. Given the magnitude of the HIV public health problem, however, it is well-addressed in many other reviews and in chapters in this issue on individual tumour viruses.

The recently-deceased pioneer in tumour virus research, George Klein, together with Ingemar Ernberg likened the field to a roller coaster [30], first going up, up and up, to become the centre piece of President Nixon's US National Cancer Institute initiative in the late 1960s, and then racing down with the newly emerging concept of cellular oncogene-tumour suppressor gene networks, most of which were discovered in studies of animal tumour viruses [9]. In the popular mind, viruses travelled from being the cause for all kinds of cancer to being irrelevant to any cancer. Klein's roller coaster has now edged up to a long and level stretch: a small number of viruses cause a large fraction of human cancer cases, particularly in less-developed nations. Studying these viruses provides new insights into all cancers and the cancer biologist who ignores molecular virology does so at his or her own professional risk.

Each human tumour virus discovery led to new general concepts in medicine and the emergence of cancer immunotherapies and vaccines show that these foreign antigen-containing tumours are low-hanging fruit that can be used as models for immunotherapeutics in non-infectious cancers. Recent studies on innate immunity and the human virome also reveal that the old notion that tumour viruses disable cell-cycle checkpoints only to replicate their own genomes is too simplistic. By better understanding how these viruses interact with the host cell, to target both replication machinery and immune pathways [31–35], we come to a more sophisticated view of the interplay between innate immunity and cellular proliferation controls [36,37]. These insights are directly relevant to cancers whether or not they are caused by a foreign virus.

2. Virus–host interaction

The human tumour viruses (table 1) range from a positively-stranded RNA virus (HCV), to a complex and stable retrovirus (HTLV-I, unlike the majority of animal tumour retroviruses that pirated cellular genes), from small (MCV and HPV) to large DNA viruses (KSHV and EBV), and include a DNA virus with a retroviral component to its life cycle (HBV). In short, they represent many forms of viruses and it initially seems difficult to describe what they have in common. From a broad perspective, however, common features begin to emerge.

(a) Viral cancers are biological accidents

None of the oncogenic viruses induce tumours as a fundamental part of their viral life cycles. All seven of the viruses cause infection and are transmitted without the majority of infected persons developing neoplasia. While an appealing hypothesis is that the growing mass of a tumour would harbour more infectious virions, this does not turn out to be the case since viruses are almost always present in a non-infectious form in tumours. It appears that viral cancers, similar to non-infectious cancers, are biological accidents and in the natural course of disease the resultant neoplasms are just as deadly to the virus as they are to their hosts. This raises an intriguing teleological question: if viral oncogenes did not evolve to cause cancer to benefit the virus, why are they conserved? For many years it was assumed that these genes simply generated an S-phase cell-cycle state to promote viral nucleic acid replication, and inhibited apoptotic routines that would be initiated from unscheduled cell-cycle entry [38]. More recent discoveries in innate immunology reveal that cells respond to infection by initiating cell-cycle arrest and initiation of programmed cell death [39]. Thus, it appears likely that viral oncogenes also serve as immune evasion genes that prevent the host cell from initiating these stereotypic responses to infection.

(b) Absence of virion production from tumour cells

Early research in tumour virology noted that a common feature of viral tumours—distinct from all other viral diseases—is that these viruses are generally ‘non-permissive’ for replication within tumour cells [40]. Active virus replication, so called lytic replication, triggers cellular host immune responses that lead to death of the infected cell generating the well-described cytopathic effect. In viral tumours, viruses are retained in each tumour cell in a near silent state (latency) either producing viral oncoproteins or initiating insertional mutagenesis that drive tumour cell proliferation—the possible exception being hepatocellular carcinoma induced by HCV. The importance of non-permissivity to tumorigenesis is seen with HPV, HBV and MCV, agents that do not typically form tumours without mutations and integration events that make it impossible for the virus to actively replicate (‘pseudolatency’). For EBV and KSHV, the viruses are in a latent state in most of the cancer cells so that no infectious particles are generated from the bulk of tumour cells. Replication occurring in a minority fraction of tumour cells can produce infectious virions but will kill the initiating tumour cells. This same principle is leveraged by viral oncolytic therapies to treat specific tumours with defects in dual immune-tumour suppressor signalling pathways that are not present in healthy surrounding tissues, making some tumours particularly susceptible to viral lytic infection and induction of an effective anticancer immune response [41].

(c) Viral cancers occur as chronic infections

All of the human cancer viruses are capable of prolonged, persistent infections. For some of these viruses, particularly EBV and MCV, infection is generally thought to be lifelong and widespread in all human populations, so much so that these viruses are part of our ‘normal’ viral flora. For HBV and HCV, it is likely that smouldering infections are responsible for chronic inflammation and cirrhosis that secondarily lead to liver cancer but these infections nonetheless typically last for decades before cancers emerge. Prolonged viral persistence increases the likelihood for required secondary conditions that change a silent viral infection into a symptomatic cancer. Kaposi's sarcoma in AIDS patients reflects loss of immunologic control over a persistent KSHV infection that usually occurred decades previously.

(d) Target cell specificity of oncogenic viruses

Neoplasia induced by tumour viruses reflect viral cell tropism. For some viruses, this is highly restricted such as HBV, which only causes primary hepatocellular carcinoma but not other cancers. Similarly, HTLV-I causes a specific subtype of CD4 + T-cell tumours called adult T-cell leukaemia (or adult T-cell leukaemia/lymphoma owing to lymphomatous lesions in the skin). In contrast, high-risk human papillomaviruses are limited to squamous epithelial cancers but these can occur at different body sites, including cervical, head-and-neck and anal cancers, that are associated with sites of sexual transmission (heterosexual, orosexual and anosexual activity, respectively). In contrast, both KSHV and EBV have a range of tissue reservoirs from which tumours can arise. KSHV is resident in both endothelial and post-germinal B cells and it causes endothelial Kaposi's sarcoma and a B-cell primary effusion lymphoma as well as a B-cell lymphoproliferative syndrome. EBV, the most ubiquitous of all the human cancer viruses, has the least restriction in the types of cancers that is causes. It is not only linked to B-cell lymphomas, but also nasopharyngeal carcinoma, stomach cancer, T-cell lymphomas, and a rare form of leiomyosarcoma (table 1).

There are certain tumours in which a tumour virus is uniformly present and required for tumorigenesis, e.g. cervical cancer and Kaposi's sarcoma. More commonly, however, viruses only cause a portion of a specific tumour histotype. While both HBV and HCV cause hepatocellular carcinoma, this cancer can also be triggered by alcoholic cirrhosis or exposure to dietary mycotoxins, such as aflatoxin mutagen [42], which can act synergistically with HBV in tumour development [43].

Differences in geographical conditions markedly affect the underlying epidemiologies of viral cancers. All cases of Burkitt's lymphoma feature chromosome translocation between c-myc on chromosome 8 and one of the immunoglobulin loci on chromosome 14 (IgG heavy chain), chromosome 2 (kappa light chain) or chromosome 22 (lambda light chain), indicative for a required cellular contribution to Burkitt's lymphomagenesis. In North American adults, including Burkitt's lymphoma in AIDS, less than 50% of tumours are EBV positive. In Papua New Guinea and sub-Saharan Africa where malaria is a risk factor for childhood Burkitt's lymphoma, however, EBV is nearly uniformly present in the tumour cells.

For Merkel cell carcinoma, both MCV-positive (approx. 80%) and MCV-negative (approx. 20%) forms exist [44]. MCV-positive tumours tend to be somewhat less aggressive and more responsive to therapy. These tumours also have no consistent pattern of cellular mutations that would suggest a cellular driver mutation, whereas MCV-negative tumours show a pattern of widespread ultraviolet light (UV)-induced genomic mutations [45]. It has been postulated that the carcinogenic load caused by an integrated and mutated MCV genomic is biologically equivalent to 10 000 s of random genomic UV mutations [46].

(e) Immune control of viral tumours

Nearly all viral cancers that have been described so far have increased incidence among immunosuppressed persons [47] (figure 1). This is particularly evident for those viruses that directly transform cells (HTLV-I, HPV, MCV, EBV and KSHV) by expression of foreign oncogenes. Thus, cancer registry studies of AIDS and transplant patients have been critical in identifying potential infectious cancers and contributed directly to discovery of tumour viruses, including KSHV [49] and MCV [50]. Furthermore, ageing is associated with more subtle immune dysfunctions and a number of tumours with viral aetiology are associated with advanced age.

Figure 1. Comparison of an infectious (Kaposi's sarcoma, KS) and a non-infectious cancer (lung and bronchus carcinoma), revealing the impact of the AIDS pandemic. These rates were collected by the US National Cancer Institute's Surveillance, Epidemiology and End-Results (SEER) cancer registry program for San Francisco County and City among males aged 20–54 years old. By 1990, epidemic KS rose approximately 240-fold over pre-AIDS era KS rates and declined in subsequent years, especially with the introduction of highly-active antiretroviral therapies in the mid-1990s. Modified from Howlader et al. [48]. (Online version in colour.)

(f) All viral cancers have non-infectious cofactors

Non-epidemiologists are sometimes perplexed by viruses as causes for cancer since the vast majority of these infections do not lead to tumours. Infectious disease specialists have long known that exposures to many infections, with exceptions such as rabies virus, smallpox virus and HIV, are often asymptomatic and therefore undetected. Non-infectious cofactors, including age, genetics, environmental factors and prior immunity, are all largely responsible for determining whether exposure to a virus leads to disease. This same principle holds true for infectious cancers. Immunodeficiency, as already described, is a common determinant for whether or not a tumour virus infection will evolve into a cancer. For some of the tumour viruses, host mutations predispose to tumour formation after infection, such as in genes encoding EVER1 and EVER2 for beta HPV-related epidermodysplasia verruciformis [51] or in SH2D1A for EBV-related X-linked lymphoproliferative disorder [52]. On the other hand, environmental exposures, such as dietary aflatoxin for HBV-related hepatocellular carcinoma [43] and malaria in childhood Burkitt's lymphoma [53], elevate risk of tumour penetrance after tumour virus exposure.

(g) Human tumour viruses versus rumour viruses

Discovery of a new suspect cancer virus is only the first step in revealing whether or not it is a cause for cancer. In some cases, such as KSHV, a causal link can be relatively quickly determined [54]. For EBV, however, over three decades passed between its discovery and the accumulation of sufficient evidence to sway the scientific community that EBV is indeed a cause of cancer [55]. Unfortunately, epidemiologic causality is often poorly equipped to establish a causal framework of a tumour virus aetiology, particularly when a tumour virus is a component of our natural viral flora [56].

Hunting for tumour viruses has given rise to many spurious claims, especially in the era of PCR—a technique that is simple to use but notorious for contamination and false positivity. SV40 is perhaps the best example of this and has been pursued as a cause for human cancers since its discovery in the early 1960s [57]. But careful analyses over decades fail to reveal any convincing evidence for SV40 playing a role in human cancer [58]. Antibody cross-reactivity, in both serologic testing and in tissue antigen detection, to known viruses and to viruses yet-to-be discovered, are also common sources for experimental mis-attribution in tumour virology [59,60].

With the advent of high-throughput sequencing, errors at the levels of the parts per billion can also lead to spurious associations. Presence of low-level HPV18 DNA sequences in The Cancer Genome Anatomy (TCGA) sequence database have been traced back to sequencing contaminating HeLa cells [61]. Sequencing-based discovery of a ‘new human hepatitis virus’ [62] actually turned out to be caused by retained RNA fragments from a marine phytoplankton virus adhering to diatomaceous earth (made from beach sand) used to make RNA isolation spin columns [63]. Endogenous retroviruses also provide rich opportunities to deceive scientists [64]: different viruses discovered in cell lines and naturally suspected to cause rhabdosarcoma and prostate cancer each represented independent examples in which endogenous retroviruses jumped from the host to a cell line when the cancer cells were passed through experimental animals [65]. Uncertainty still exists as to whether Peyton Rous discovered a pre-existing virus that caused the original chicken tumour or if this agent was accidentally and secondarily amplified by the experimental transplantation techniques [6,39] that Rous used to isolate the transforming virus.

New technologies promise to uncover new tumour virus suspects but will also provide opportunities for mistakes and scientific confusion. Simple scientific rigour and a healthy level of scepticism remain among the most important tools for any tumour virologist.

(h) Viral cancers are predominantly a public health burden in developing countries

While wealthy nations in Europe and North America generally have low viral cancer burdens, infection can cause over one-half of cancers in some developing countries. The types of cancer in developing countries, however, vary widely and are influenced by concurrent patterns of AIDS immunosuppression. Among persons from sub-Saharan Africa, who have historically had high endemic KSHV infection prevalence, the HIV pandemic led to an epidemic of AIDS-KS [66]. Other KSHV-endemic areas include western China [67] and Andean South America. In contrast, KSHV and KS are relatively rare for most Southeastern Asians but these same populations have high rates of EBV-related nasopharyngeal and stomach cancers [23] and HBV-related hepatocellular carcinoma [68].

3. Pathways to discovery and detection of human oncogenic viruses

(a) Epidemiology

The pattern of incidence of certain types of tumour may provide an indication that a tumour has an infection as part of its aetiology. The unusual geographical distribution of Burkitt's lymphoma described by Dennis Burkitt led Sir Anthony Epstein in turn to search for a virus. In fact, the causative virus that he discovered [69] is nearly ubiquitous worldwide and the unique geographical distribution of Burkitt's lymphoma is more closely related to associated environmental factors such as malaria prevalence discussed above. Likewise, place of birth studies for patients with adult T-cell leukaemia in Japan led Takatsuki and colleagues [70] to suggest that there may be a transmissible component which after the discovery of HTLV-1 in USA [71] turned out to be that virus [72].

Given the dependence of viral tumour expression on immune suppression, epidemiologic studies of AIDS and transplant patients have been a rich source for identifying new infectious cancers. Analysis of US AIDS patient data by Beral et al. [49] provided a detailed epidemiologic description of the likely ‘KS agent’ and led directly to the discovery of KSHV using genomic subtractive methods [73]. Similarly, elevated rates of Merkel cell carcinoma among AIDS patients compared to the general population [50] provided the basis for a transcriptomic search that identified Merkel cell polyomavirus [74]. The natural history of chronic viral hepatitis preceding onset of hepatocellular carcinoma was a key feature in the identification of HBV and HCV as causes for liver cancer [75,76].

(b) Detection of virus particles

Epstein, Achong and Barr discovered the virus causing Burkitt's lymphoma by electron microscopy (EM) [69], but for reasons already described, this is the least promising pathway to virus detection. Poiesz et al. [71] searched for reverse transcriptase activity in the culture supernatants of a cutaneous T-cell lymphoma, now recognized to be a form of adult T-cell leukaemia, and then confirmed the presence of type C retroviral particles (HTLV-1) by electron microscopy.

(c) Detection of viral antigens and antibodies

Markers for virus infection have sometimes been discovered before the viruses themselves and helped to lead to their discovery. The classic example is Baruch Blumberg's delineation of Australia antigen in the 1960s which he first interpreted as a genetic blood group. Later it was discovered by Harvey Alter and Blumberg, and also by Alfred Prince, to be the surface antigen of HBV [77,78]. Similarly, a link between nasopharyngeal carcinoma and EBV was first found by serologic studies [79]. As Louis Pasteur famously observed, chance favours the prepared mind, and these investigators soon realized the significance of their unexpected results.

(d) Viral nucleic acid detection

The non-permissivity of tumour viruses led Harald zur Hausen to focus on identifying viral nucleic acids in tumour tissues rather than encapsidated virions [80]. Knowing that genital wart HPV type 6 had similar epidemiologic features to cervical cancer, he and his colleagues used ‘de-tuned’ Southern hybridization with HPV-6 DNA to isolate cross-reactive HPV-16 DNA from a cervical tumour [7] and then HPV-18 from tumour and HeLa cells [81]. With this breakthrough, it became apparent that discovering tumour viruses was more a matter of gene hunting rather than virus hunting.

As with discovery of high-risk HPV, careful epidemiologic studies would be central to subsequent tumour virus discoveries. By 1989, it was clear that a substantial fraction of chronic hepatitis cases had a nonA and nonB viral origin that could be transmitted in primate animal models. Houghton and colleagues [82] analysed a chimpanzee that had been infected with human nonA, nonB hepatitis serum and generated a cDNA library from its plasma. Using well-characterized human sera from a nonA, nonB case, they searched for cDNA clones that when expressed were specifically reactive to the sera and they succeeded in isolating RNA fragments of hepatitis C virus, which quickly led to full-length viral genome cloning.

Identification of Kaposi's sarcoma herpesvirus (KSHV) in 1994 also depended on isolating cancer-associated genes belonging to a new virus. Applying subtractive representational difference analysis [83] to Kaposi's sarcoma tumours, Chang and colleagues isolated herpesvirus DNA fragments from the tumour which were not present in healthy tissues from the same patient [73]. This new human herpesvirus (KSHV or HHV8) was rapidly shown to fulfil the epidemiologic predictions made for the ‘KS agent’ and also to cause primary effusion lymphoma [84] and multicentric Castleman's disease [85].

With completion of the human genome and advent of inexpensive sequencing technologies, zur Hausen's strategy for tumour virus discovery could be directly approached. Based on the assumption that any directly-transforming tumour virus will express foreign oncoprotein-encoding mRNA in each tumour cell, Feng et al. [86] developed a method to computationally subtract known human sequences from a tumour transcriptome (called digital transcriptome subtraction), which was used to detect Merkel cell polyomavirus DNA from a Merkel cell tumour [74].

The decade following discovery of MCV witnessed an explosion in cancer genomic sequencing, as described by Tang and Larsson Lekholm in this issue [16], and yet no new cancer virus suspects have been uncovered. Time will tell whether we have exhausted the repertoire of human cancer viruses or if there are additional suspects that have been missed. Perhaps, some cancers harbour an agent that is so distant from known viruses that we cannot distinguish it from junk sequences that are in the discard folders on sequencing computers. But MCV also showed that we live with a complex and largely hidden virome that contributes to our health, by training portions of our immune system [87], or can cause cancer when a floral virus undergoes a precise set of mutations [88]. This issue of the Philosophical Transactions explores recent discoveries in human tumour virology, how these cancers can be prevented and controlled, and suggests that Klein's roller coaster may be headed up again.

Data accessibility

This article has no additional data.

Competing interests

We declare we have no competing interests.

Funding

This work was supported by U.S. National Institutes of Health Grants CA136363, CA120726 and CA170354 (to Y.C. and P.S.M.) and in part by Award P30CA047904 to the University of Pittsburgh Cancer Institute. Y.C. and P.S.M. are also supported by the American Cancer Society, the Pittsburgh Foundation and the UPMC Foundation.


Overview of Viruses

Viruses are the smallest parasites, typically ranging from 0.02 to 0.3 micrometer, although several very large viruses up to 1 micrometer long (megavirus, pandoravirus) have recently been discovered. Viruses depend completely on cells (bacterial, plant, or animal) to reproduce. Viruses have an outer cover of protein and sometimes lipid, an RNA or DNA core, and sometimes enzymes needed for the first steps of viral replication.

Viruses are classified principally according to the nature and structure of their genome and their method of replication, not according to the diseases they cause. Thus, there are DNA viruses and RNA viruses each type may have single or double strands of genetic material. Single-strand RNA viruses are further divided into those with (+) sense and (-) sense RNA. DNA viruses typically replicate in the host cell nucleus, and RNA viruses typically replicate in the cytoplasm. However, certain single-strand, (+) sense RNA viruses termed retroviruses use a very different method of replication.

Retroviruses use reverse transcription to create a double-stranded DNA copy (a provirus) of their RNA genome, which is inserted into the genome of their host cell. Reverse transcription is accomplished using the enzyme reverse transcriptase, which the virus carries with it inside its shell. Examples of retroviruses are the human immunodeficiency viruses and the human T-cell leukemia viruses. Once the provirus is integrated into the host cell DNA, it is transcribed using typical cellular mechanisms to produce viral proteins and genetic material. If the infected cell belongs to the germline, the integrated provirus can become established as an endogenous retrovirus that is transmitted to offspring.

The sequencing of the human genome revealed that at least 1% of the human genome consists of endogenous retroviral sequences, representing past encounters with retroviruses during the course of human evolution. A few endogenous human retroviruses have remained transcriptionally active and produce functional proteins (eg, the syncytins that contribute to the structure of the human placenta). Some experts speculate that some disorders of uncertain etiology, such as multiple sclerosis, certain autoimmune disorders, and various cancers, may be caused by endogenous retroviruses.

Because RNA transcription does not involve the same error-checking mechanisms as DNA transcription, RNA viruses, particularly retroviruses, are particularly prone to mutation.

For infection to occur, the virus first attaches to the host cell at one or one of several receptor molecules on the cell surface. The viral DNA or RNA then enters the host cell and separates from the outer cover (uncoating) and replicates inside the host cell in a process that requires specific enzymes. The newly synthesized viral components then assemble into a complete virus particle. The host cell typically dies, releasing new viruses that infect other host cells. Each step of viral replication involves different enzymes and substrates and offers an opportunity to interfere with the process of infection.

The consequences of viral infection vary considerably. Many infections cause acute illness after a brief incubation period, but some are asymptomatic or cause minor symptoms that may not be recognized except in retrospect. Many viral infections are cleared by the body’s defenses, but some remain in a latent state, and some cause chronic disease.

In latent infection, viral RNA or DNA remains in host cells but does not replicate or cause disease for a long time, sometimes for many years. Latent viral infections may be transmissible during the asymptomatic period, facilitating person-to-person spread. Sometimes a trigger (particularly immunosuppression) causes reactivation.


Temin, H. M., Virology, 23, 486 (1964).

Temin, H. M., Cancer Res., 28, 1835 (1968).

Temin, H. M., and Mizutani, S., Nature, 226, 1211 (1970).

Baltimore, D., Nature, 226, 1209 (1970).

Temin, H. M., J. Nat. Cancer Inst. (editorial), 46, III (1971).

Temin, H. M., Persp. Biol. Med., 14, 11 (1970).

Gallo, R. C., Blood (editorial) (in the press).

Culliton, B. J., Science, 172, 9 (1971).

Solomon, J., The Sciences, 2, 6 (1971).

Spiegelman, S., Burny, A., Das, M. R., Keydar, J., Schlom, J., Travnicek, M., and Watson, K., Nature, 227, 563 (1970).

Spiegelman, S., Burny, A., Das, M. R., Keydar, J., Schlom, J., Travnicek, M., and Watson, K., Nature, 228, 430 (1970).

Schlom, J., Harter, D. H., Burny, A., and Spiegelman, S., Proc. US Nat. Acad. Sci., 68, 182 (1971).

Temin, H. M., Virology, 23, 486 (1964).

Bader, J. P., Virology, 22, 462 (1964).

Baluda, M. B., and Nayak, D. P., J. Virol., 4, 554 (1969).

Temin, H. M., Cancer. Res., 28, 1835 (1968).

Duesberg, P. H., Vogt, P. K., and Canaani, E., in Second Lepetit Colloquium on Biology and Medicine, The Biology of Oncogenic Viruses (edit. by Silvestri, L.) (North-Holland, Amsterdam, 1971).

Hanafusa, H., and Hanafusa, T., Virology, 43, 313 (1971).

Robinson, W. S., and Robinson, H. L., Virology, 44, 457 (1971).

Gurgo, D., Ray, R. K., Thiry, L., and Green, M., Nature, 229, 111 (1971).

Gallo, R. C., Yang, S. S., and Ting, R. C., Nature, 228, 927 (1970).

Gallo, R. C., Yang, S. S., Smith, R. G., Herrera, F., Ting, R. C., Bobrow, S. N., Davis, C., and Fujioka, S., in Second Lepetit Colloquium on Biology and Medicine, The Biology of Oncogenic Viruses (edit. by Silvestri, L.) (North-Holland, Amsterdam, 1971).

Yang, S. S., Smith, R. G., Ting, R. C., and Gallo, R. C., J. Nat. Cancer Inst. (in the press).

Brockman, W. W., Carter, W., Li, L. H., Reusser, F., and Nichol, F. R., Nature, 230, 249 (1971).

Carter, W. A., Brockman, W. W., and Borden, E. C., Nature New Biology, 232, 212 (1971).

Parkman, R., Levy, J. A., and Ting, R. C., Science, 168, 387 (1970).

Spiegelman, S., Burny, A., Das, M. R., Keydar, J., Schlom, J., Travnicek, M., and Watson, K., Nature, 227, 563 (1970).

Duesberg, P. H., and Canaani, E., Virology, 42, 783 (1970).

Mizutani, S., Boettiger, D., and Temin, H. M., Nature, 228, 424 (1970).

Kacian, D. L., Watson, K. F., Burny, A., and Spiegelman, S., Biochim. Biophys. Acta, 246, 365 (1971).

Duesberg, P., Helm, K. V. D., and Canaani, E., Proc. US Nat. Acad. Sci., 68, 747 (1971).

Ross, J., Scolnick, E. M., Todaro, G. J., and Aaronson, S. A., Nature New Biology, 231, 163 (1971).

Duesberg, P., Helm, K. V. D., and Canaani, E., Proc. US Nat. Acad. Sci., 68, 2505 (1971).

Baltimore, D., and Smoler, D., Proc. US Nat. Acad. Sci., 68, 1507 (1971).

Verma, I. M., Meuth, N. L., Bromfeld, E., Manly, K. F., and Baltimore, D., Nature New Biology, 233, 131 (1971).

Schlom, J., Spiegelman, S., and Moore, D., Nature, 231, 97 (1971).

Priori, E. S., Dmochowski, L., Myers, B., and Wilbur, J. R., Nature New Biology, 232, 61 (1971).

Gallo, R., Sarin, P., Allen, P., Newton, B., Bowen, J., Priori, E. S., and Dmochowski, L., Nature New Biology, 232, 140 (1971).

Wade, N., Science, 173, 1220 (1971).

Gerwin, B. I., Todaro, G., Zeve, V., Scolnick, E. M., and Aaronson, S. A., Nature, 228, 435 (1971).

Fujinaga, K., Parsons, J. T., Beard, J. W., Beard, D., and Green, M., Proc. US Nat. Acad. Sci., 67, 1432 (1970).

Spiegelman, S., Presented at the Second Colloquium on Biology and Medicine, Paris (1970).

Goodman, N. C., and Spiegelman, S., Proc. US Nat. Acad. Sci., 68, 2203 (1971).

Scolnick, E. M., Aaronson, S. A., Todaro, G. J., and Parks, W. P., Nature, 229, 318 (1971).

Aaronson, S., Parks, W. P., Scolnick, E. M., and Todaro, G. J., Proc. US Nat. Acad. Sci., 68, 920 (1971).

Scolnick, E. M., Presented at the Second International Congress of Virology, Budapest (1971).

Todaro, G., in US National Academy of Science Symposium (in the press).

Gallo, R. C., Yang, S. S., Smith, R. G., Herrera, F., Ting, R. C., and Fujioka, S., in Nucleic Acid-Protein Interactions and Nucleic Acid Synthesis in Viral Infection, Miami Winter Symposium (edit. by Ribbons, D. W., Woessner, J. F., and Schultz, J.) (North-Holland, Amsterdam, 1971).

Hirschman, S. Z., Science, 173, 441 (1971).

Müller, W. E. G., Zahn, R. K., and Seidal, H. J., Nature New Biology, 232, 143 (1971).

Tuominen, F. W., and Kenney, F. T., Proc. US Nat. Acad. Sci., 68, 2198 (1971).

Abrell, J., Smith, R. G., Robert, M., and Gallo, R. C. (unpublished results).

Yang, S. S., Ting, R. C., and Gallo, R. C., Proc. Amer. Assoc. Cancer Res., 12, 36 (1971).

Spiegelman, S., Presented at the Miami Winter Symposium on Nucleic Acid-Protein Interactions and Nucleic Acid Synthesis in Viral Infection (1971).

Lee-Huang, S., and Cavalieri, L. F., Proc. US Nat. Acad. Sci., 50, 1116 (1963).

Kiessling, A. A., Weber, G. H., Deeney, A. O., Possehl, E. A., and Beaudreau, G. S., J. Virol., 7, 221 (1971).

Penner, P. E., Cohen, L. H., and Loeb, L. A., Nature New Biology, 232, 58 (1971).

Müller, W. E. G., Yamazaki, Z., Zahn, R. K., Brehm, G., and Korting, G., Biochem. Biophys. Res. Commun., 44, 433 (1971).

Penner, P. E., Cohen, L. H., and Loeb, L. A., Biochem. Biophys. Res. Commun., 42, 1228 (1971).

Cavalieri, L. F., and Carroll, E., Biochem. Biophys. Res. Commun., 41, 1055 (1971).

Coffin, J., and Temin, H. M., J. Virol. (in the press).

Ackermann, W. W., Murphy, W. H., Miller, B. A., Kurtz, H., and Barker, S. T., Biochem. Biophys. Res. Commun., 42, 723 (1971).


Discovery of oncogenes: The advent of molecular cancer research

In their classic paper on the identification of the transforming principle of Rous sarcoma virus (RSV) published 1970 in PNAS (1), Peter Duesberg at the University of California, Berkeley, and Peter Vogt, then at the University of Washington, Seattle, drew a seemingly simple yet groundbreaking conclusion. When they analyzed the genomic RNAs of transforming, acutely oncogenic RSV and of transformation-defective (td) mutant derivatives, they found that all transforming virus stocks contained two classes of RNA subunits, a larger one (a) and a smaller one (b), whereas the nontransforming yet replication-competent mutants contained the smaller b subunits only. Duesberg and Vogt concluded that the larger a subunit contained the transforming principle of RSV. Based on this and on subsequent structural comparisons of the a and b subunits of biologically cloned viruses, the transforming principle was defined by the remarkably simple equation ab = x and was later termed src (for sarcoma). The first biochemical identification of a cancer gene was achieved, initially in a chicken virus. However, the principal proof of a physical underpinning of the cancer gene hypothesis had tremendous impact on a fundamental challenge of medicine, decoding the molecular basis of human carcinogenesis.

The genetic and biochemical investigations of the chicken tumor virus RSV and the persistent search for its transforming principle are a classic paradigm in cellular and molecular cancer research (2, 3). In 1911, Peyton Rous at the Rockefeller Institute in New York discovered the first virus—later termed RSV—that could induce solid tumors in infected fowl, demonstrated by experimental transmission of sarcomas using cell-free filtrates of tumor extracts (4). This seminal discovery started the field of tumor virology (2, 3, 5). However, almost half a century had to pass before the first quantitative biological tools were developed to study the biology of RSV and its interaction with infected cells in detail. RSV is capable of transforming primary chicken embryo fibroblasts in culture, and the focus assay developed in 1958 by Howard Temin and Harry Rubin at the California Institute of Technology allowed a quantitative assessment of the virus–cell interaction leading to malignant cell transformation (6). The next crucial steps toward the identification of the underlying principle of RSV oncogenicity were based on classic genetics. The characterization of various viral strains that induced different morphologies of transformed cells suggested that the phenotype of the cancer cell is controlled by the incoming genetic information carried by the viral genome. The isolation of RSV mutants that can transform cells but do not produce infectious progeny, or vice versa, can replicate but have lost cell transforming capacity, demonstrated that viral replication and oncogenicity are genetically separable, independent functions of RSV (2, 3). A groundbreaking leap forward came from studies of conditional mutants (7, 8). In 1970, Steve Martin at the University of California, Berkeley isolated a temperature-sensitive mutant of RSV that did not transform cells at the nonpermissive temperature but replicated normally, indicating the existence of a viral gene that is necessary for cell transformation but dispensable for replication (8).

In the same year, a marvelous synergistic effort of biochemistry and virus genetics led to the first physical identification of an oncogene, reported in the classic paper by Duesberg and Vogt in PNAS (1). Their biochemical approach in the hunt for the transforming principle made use of the availability of td deletion mutants of RSV and of nontransforming viruses associated with avian sarcoma or leukemia viruses (2). In essence, the experimental design involved coelectrophoresis of viral RNAs from transforming and nontransforming avian retroviruses, including various strains of RSV and td or associated viruses. Notably, current nucleic acid technologies, like reverse transcription, blotting, cloning, or sequencing, were not yet established in those days. All of this had to be done by metabolic labeling of infected cell cultures with radiolabeled precursors ([ 3 H]uridine or [ 32 P]H3PO4), purification of viruses, extraction of viral RNA, polyacrylamide gel electrophoresis, gel slicing, and scintillation counting of 1-mm gel slices. The results were as clear as compelling. After heat dissociation, the 60–70S RNA complexes of all viruses able to transform chicken embryo fibroblasts resolved into two types of RNA species at variable ratios: a large a subunit and a smaller b subunit (Fig. 1). Nontransforming yet replication-competent viruses always contained b subunits only. It was concluded that the presence of genetic material in the a subunit, which is absent from the b subunit, is responsible for the oncogenic capacity of RSV. Final proof that a and b are structurally related by the equation a = b + x, and that x is indeed a contiguous segment near the 3′ end of RSV RNA (Fig. 1), came from comparative mapping of the genomes of transforming viruses, their td derivatives, and other gene-specific deletion mutants. The biochemical mapping used 2D electrophoresis-homochromatography of 32 P-labeled RNase T1-resistant oligonucleotides and was done in collaborations of the Duesberg laboratory with Peter Vogt, then at the University of Southern California, Los Angeles, and with Hidesaburo Hanafusa at the Rockefeller University (9 ⇓ –11).

Biochemical definition of src, the first oncogene. Panels A and B, above, are from the original PNAS paper by Duesberg and Vogt (1). They show electropherograms of the 60–70S RNAs from two transforming strains of RSV, Schmidt-Ruppin (SR) and B77, before (A) and after (B) heat-dissociation. Insets in A show the final sucrose gradient purification of the RNAs before electrophoretic analysis. The heat-dissociated RNAs were resolved into two subunits with lower (a) and higher (b) electrophoretic mobility. Analyses of biologically cloned viruses revealed that the larger subunit represents the genomic RNA of transforming RSV, whereas the b subunit is the genome of transformation-defective (td) mutants spontaneously segregating from RSV (1, 10). Subsequent mapping studies (10, 11) confirmed that the genomes of RSV and of td mutants share all replicative genes (gag, pol, env) and that the size difference (ab = x) is caused by the additional src gene at the 3′ end of the RSV genome. Cells infected by RSV become transformed (indicated by rounding) and produce virus progeny (red star symbols), whereas td RSV replicates (green star symbols) but does not transform the host cell. A and B reproduced with permission from ref. 1.

In 1976, pioneering experiments performed in the laboratory of Harold Varmus and Mike Bishop at the University of California, San Francisco, in collaboration with Peter Vogt at the University of Southern California, changed the whole field of tumor virology and cancer genetics (12). Their finding—that the src gene of RSV (v-src) is in fact a transduced allele of a cellular gene (c-src) picked up by recombination during the retroviral life cycle—is one of the most influential discoveries in cancer research. It immediately converted the purely virological matter of oncogenes to a cellular one, relevant for all animals and man, as was quickly shown by the identification of c-src in many species. Principally, any activating mutation or deregulation of cellular oncogenes, also termed proto-oncogenes in their normal nonmutated form, could now lead to cancer, with or without viral involvement. The experimental design for the discovery of c-src exploited the availability of reverse transcription and the definition of v-src by the size difference (ab = x) of transforming and td RSV genomes (Fig. 1 and see above). Synthesis of RSV cDNA and subtractive hybridization with td RNA led to a src-specific DNA probe that was used for annealing experiments showing that normal cells contain sequences closely related to src in their genomic DNA (12). Subsequent reports on the experimental recovery of transforming viruses by recombination of td RSV carrying partial v-src deletions with cellular sequences corroborated the close v-src/c-src relationship (13). For the landmark discovery of the cellular origin of retroviral oncogenes, Bishop and Varmus were awarded the Nobel Prize in Physiology or Medicine in 1989. Following the identification of v-src and c-src, the immunological detection and characterization of the src protein product as a tyrosine-specific protein kinase with modular protein interaction domains were further groundbreaking discoveries (14 ⇓ –16). Moreover, the src paradigm immediately stimulated the search for the transforming principle of other highly oncogenic avian retroviruses. In two studies from the Vogt and Duesberg laboratories, also published in PNAS in 1977 and 1979, analyses of the genomes of avian acute leukemia viruses MC29 and avian erythroblastosis virus, using the biochemical approach described above, led to the discovery of specific sequences unrelated to replicative genes or to the prototypic src oncogene (17, 18). These novel oncogenes were later shown to be derived from cellular oncogenes, which today are known as major drivers of human cancer, MYC, and the ERBB/EGFR gene, respectively (2). While src, myc, and erbB were originally discovered in avian tumor viruses (2, 19), other prominent oncogenes, like ras, were identified in murine tumor viruses or in independent seminal experiments by direct transfection of human tumor cell DNA into recipient cells (2, 20).

Having spent postdoctoral time both in the Vogt and Duesberg laboratories right at the time when all of this was happening, I can vividly recall the exciting, almost adventurous spirit of the oncogene discovery days. Particularly stimulating were the joint informal meetings of the Vogt, Duesberg, and Bishop/Varmus groups held at alternating California laboratory sites, where ideas, strategies, and results were freely exchanged and crucial collaborations initiated. From the pioneering discovery of the first oncogene in a chicken virus, oncogene research has developed into a central topic in human cancer genetics. Several oncogenes originally identified in retroviruses are now recognized as major drivers in human cancers, and drugs targeted at specific oncogene functions are used in cancer therapy (2). Furthermore, many proto-oncogenes are essential genes involved in fundamental processes in normal cells, like growth, metabolism, or differentiation. Oncogenes and proto-oncogenes will remain in the focus of biology, biochemistry, and medicine.


Discussion

NMD plays a well-documented role in regulating the abundance of a large variety of cellular transcripts however, our knowledge of the interplay between NMD and viral infection, in particular infection with DNA viruses, remains rudimentary. Along these lines, although recent reports have revealed a contribution of NMD to RNA virus infections, only few bona fide viral NMD targets have been identified [21–27]. In this study, we show that NMD targets the spliced, polycistronic EBV and KSHV transactivator-encoding transcripts for degradation through the recognition of NMD-inducing features in their 3′ UTRs this ultimately keeps the abundance of the EBV and KSHV Rta proteins to a minimum, thereby suppressing virus reactivation. Our findings thus identify NMD as a key regulator of oncogenic DNA virus infection.

The biphasic life cycle consisting of latency establishment in long-lived cells and the occasional reactivation that results in production of viral progeny is a hallmark of herpesvirus infection. Given the central role of the Rta transactivator proteins of EBV and KSHV in initiating lytic reactivation, it is not surprising that their expression is extensively regulated. The cotranslational regulation of Rta mRNA stability by NMD that we identified here provides an extra layer of control in addition to the epigenetic, transcriptional, and posttranscriptional regulatory mechanisms that have been reported previously [37,48–50]. Mechanistically, our results suggest that NMD prevents viral reactivation by degrading transactivator transcripts that are produced at low levels in latently infected cells. The strong lytic reactivation that we observed upon inhibition of NMD, to a similar extent as treatment with potent chemical inducers such as sodium butyrate, underscores the importance of NMD in preserving EBV and KSHV latency. Future studies will need to determine the exact interplay and temporal sequence of the various regulatory mechanisms that modulate EBV and KSHV reactivation.

Our results also indicated that the NMD-mediated degradation of EBV BRLF1-encoding transcripts depends on the presence of introns as well as the long 3′ UTR. Our results are consistent with the findings by Zhao and colleagues who recently reported that the 3′ UTR of KSHV Orf50 transcripts is recognized by the NMD machinery in an intron-dependent manner, and that, consequently, silencing of the NMD components UPF1 or UPF3X resulted in de-repression of KSHV reactivation [51]. The conservation of these NMD-inducing features in the transactivator locus of EBV and KSHV may suggest that these viruses have adopted NMD-mediated regulation of viral gene expression and reactivation as an integral part of their life cycle. Besides the transactivator-encoding transcripts that we identified as NMD targets, gammaherpesvirus genomes also encode many other spliced and/or polycistronic transcripts that often display features known to induce NMD-mediated degradation [28–31]. Several of these transcripts were found to be associated with UPF1 in our RIP-seq analysis. Future studies will need to investigate whether other viral transcripts are targeted by the NMD machinery, which would further expand the role of NMD in regulating EBV and KSHV gene expression. Furthermore, the Gene Set Enrichment Analysis (GSEA) of UPF1-associated cellular transcripts showed that 14 gene sets were significantly enriched with UPF1 or phospho-UPF1 in EBV-infected cells, suggesting that NMD may also modulate the activity of several cellular processes during infection (S8 Fig). It will be interesting to determine the extent to which NMD-mediated control of these pathways regulates EBV and KSHV reactivation, especially because some of these pathways have previously been implicated in the regulation of EBV and/or KSHV reactivation these include the unfolded protein response, whose regulation by NMD was recently shown to affect KSHV reactivation [51] and the MYC pathway that was recently reported to suppress EBV reactivation [52]. In view of this, it seems plausible that EBV and KSHV have evolved mechanisms to manipulate NMD-mediated transcript degradation. While to date these functions have not been identified for herpesviruses, several positive-sense RNA viruses as well as retroviruses have recently been shown to compromise NMD activity by interfering with the function of specific NMD factors furthermore, certain cellular transcripts escape NMD by encoding specific features that inhibit NMD-mediated degradation [22,23,25,27,53–55]. Taken together, we anticipate future studies to reveal a more intricate and dynamic interplay between gammaherpesvirus infection and the host NMD pathway.

The balance between latent and lytic infection is a significant determinant in EBV- and KSHV-associated tumorigenesis [12,13]. Given the important role of NMD in regulating the latent-to-lytic switch, it will be important to determine how changes in NMD activity by host regulatory mechanisms or environmental triggers affect gammaherpesvirus replication and oncogenesis. For example, reported variations in intrinsic NMD activity between cells may contribute to gammaherpesvirus tissue and cell tropism, and although we observed robust EBV reactivation by NMD inhibition in all cell types tested, there might still be differences in NMD-mediated control of Rta expression and/or cellular pathways in epithelial versus lymphoid cells [56]. Finally, besides cell type–specific differences, variations in NMD efficiency between individuals, which have been shown to affect the outcome of certain genetic diseases, may affect susceptibility to EBV- and KSHV-associated diseases [57].

Since the majority of cells in EBV- and KSHV-associated cancers are latently infected, therapeutic induction of lytic reactivation can be used to specifically sensitize tumor cells to antiviral drugs and to activate cytotoxic T-lymphocyte responses that are typically directed against lytic antigens [12,48,58,59]. In this study, we observed a strong induction of EBV and KSHV reactivation by the small-molecule NMD-inhibitor NMDI-1 in a variety of cell types, even those that are notoriously refractory to reactivation. Concentrations of NMDI-1 below those used in our study have successfully been used in in vivo studies without apparent toxicity [60,61]. Moreover, it was recently reported that modest NMD inhibition does not have an appreciable negative impact on overall health in mice [62]. Together, this suggests that NMD inhibition may be a potential strategy to therapeutically induce viral reactivation in the treatment of EBV- and KSHV-associated malignancies.

In conclusion, our study identifies the cellular NMD pathway as a prominent regulator of EBV and KSHV reactivation, providing novel insight into the intricate virus–host interactions that play a fundamental role in viral disease progression.


Methods

Subjects and sequence data

We analyzed mammalian DNA virus sequences in metagenomic, whole genome shotgun sequence (WGS) data sets from 706 samples, which were produced on the Illumina platform (Illumina, Inc., San Diego, California, United States) by the HMP (Additional file 1: Table S1) [2]. Our analysis was limited to DNA viruses because RNA was not isolated from the samples in the HMP study. Furthermore, sequencing library construction protocols are designed for dsDNA therefore, our analysis focused on dsDNA viruses and dsDNA replicative intermediates of ssDNA viruses. The sampled sites included in the WGS data set were nose (anterior nares), skin (retroauricular crease), mouth (buccal mucosa, tongue dorsum, subgingival plaque, supragingival plaque, and throat), vagina (primarily posterior fornix, but a few samples from vaginal introitus and mid-vagina were also included), and gut (stool). In some cases, multiple (usually two) visits from the same individual were included. Samples were collected according to standardized protocols [36]. Clinicians collected samples from each anatomic site using Catch-All™ Sample Collection Swabs, with the exception of the stool sample, which was self-collected. DNA was extracted from each sample using the MoBio PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, California, United States). The Illumina GAIIX platform was used to generate 100 bp paired-end reads, with the target of generating 10 Gb sequence per sample. BMTagger was used to identify human sequences, which were subsequently removed from the data set. Duplicate, low quality, and low complexity sequences were also trimmed or removed.

Clinical data from this study were jointly produced by the Baylor College of Medicine and the Washington University School of Medicine. Sequencing data were produced by the Baylor College of Medicine Human Genome Sequencing Center, The Broad Institute, the Genome Institute at Washington University, and the J. Craig Venter Institute. The metadata were submitted by the EMMES Corporation, which serves as the clinical data collection site for the HMP. Only a subset of samples collected by the HMP was subjected to WGS. Subjects were sampled using a protocol that was approved by the Institutional Review Board for Baylor College of Medicine and the Human Research Subjects Protection Office at Washington University School of Medicine, and written informed consent was obtained from all subjects [2],[37]. Before they were admitted to the study or sampled, subjects were required to meet an extensive set of clinical criteria, which established them as generally healthy adults. Importantly, entry criteria established that the subjects were not symptomatic for acute infections, had not been diagnosed with HPV infection within the last two years, and had not had any active genital herpes infection within the last two months (females) [37]. Subjects were sampled up to three times at the same body sites, with visits separated by 30 to 359 days. The WGS data include samples from 102 young adults (18 to 40 years old), of whom 46 were female. Sample collection was divide across two locales, with 46% collected at Baylor College of Medicine in Houston, Texas, and 54% at Washington University in St Louis. Our study population comprised 86% Caucasian, 6% Black, and 8% Asian participants.

Identification of viral sequences

We began analysis with the human-screened, processed data sets provided by the HMP [2]. Viral sequences were identified based on similarity to virus reference genomes. We optimized the analysis pipeline for viral sequence detection by increasing the sensitivity of the alignments. This was done by allowing mismatches between the query and reference and by including both nucleotide and translated sequence alignments so sequences that were divergent from reference genomes would be detected. This analysis is an improved version of the pipeline we described previously [35]. A brief description of the pipeline follows. First, sequence reads were aligned against a virus reference database using a tool for nucleotide sequence alignment. In this version of the pipeline, a Real Time Genomics map (Real Time Genomics, Hamilton, New Zealand) was used to align sequences to the reference sequence database. The following parameters allowed us to identify sequences with nucleotide sequence similarity to viral reference sequences: −-repeat-freq 97% -e 10% –w 15 –n 255 –penalize-unknowns. The sequences in the reference sequence database included all of the sequences classified as viral in the National Center for Biotechnology Information Nucleotide database [38], which were found by using the search term `virus’. This included viral genomes and partial viral sequences. Next, sequences that were not aligned were subjected to translated sequence alignments to the same viral references, which were translated in six frames. This version of the pipeline used MBLASTX software (MulticoreWare Sunnyvale, California, United States) [39] with the following parameters: −m 30 –e 1e-02. After this initial screen to identify sequences with similarity to viral genomes, the subset of sequences identified was aligned to the larger nucleotide and translated amino acid sequence databases [40], which include entries from a more comprehensive set of organisms. Again, this version of the pipeline used Real Time Genomics mapping and MBLASTX with the parameters described above. Finally, sequences that unambiguously aligned to viral references in the larger databases were considered viral and included in the downstream analysis, and sequences that could not be clearly classified, such as repetitive sequences, were disregarded. This is a conservative method for identifying viral sequences within the samples. The number of sequences aligned to mammalian DNA virus genera are shown in Additional file 1: Table S2. Endogenous retroviruses integrated into the human genome were excluded from analysis because many endogenous retrovirus sequences were removed during the human screening step carried out by the HMP Consortium, and, therefore, the endogenous retrovirus sequence counts obtained from our pipeline are incomplete. Virus sequences were classified at the genus level, and species level classifications were determined after manual review.

Correlation of viruses with bacterial communities

The characterization of bacterial communities by the HMP was used to correlate viral and bacterial community structure in the vaginal samples [2],[41]. Relative abundance of the bacterial communities was calculated by taking the (depth of coverage × 100 Mb/number of covered bases). Several small, incomplete references, which included ribosomal sequences, had been included in the HMP reference database. These references were removed from the report by excluding references smaller than 100,000 bases in length in the final report of organisms present in the vaginal samples. Patient data were obtained through the Database of Genotypes and Phenotypes (study accession phs000228.v2.p1 [42]). Samples were clustered based on Bray-Curtis dissimilarity of the bacterial community structure and visualized using iTOL [43].


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Innate Immunity

Although adaptive immunity seems to provide and, in fact, represent even the major mode of anticancer action for OVs, it is also evident that an initial host response against an administered OV could destroy it along with the infected cells before the OV has a chance to replicate and induce cytotoxicity of a magnitude that is sufficient to set up an effective vaccination response (17). Location and site of OV administration is an important determinant of the characteristics of these initial host responses against the OV. For instance, intravenous or intra-arterial administration of OVs, such as recombinant HSV1, leads to its rapid recognition and elimination by the circulating complement and antibodies of the humoral defense system (18, 19). This has also been shown for VV (20), NDV (21), MV (22), and Ad (23, 24). Intratumoral administration can also lead to complement- and antibody-mediated destruction of the OV. In addition, intracellular and microenvironmental antiviral defense responses in infected tumor cells can also greatly limit the magnitude of OV replication (25–31). Finally, innate immune cells can rapidly respond to an administered OV, further limiting its survival and that of OV-infected tumor cells (32–35). In all these models, circumvention of such responses using pharmacologic agents, such as histone deacetylase (HDAC) inhibitors or immunomodulating drugs, or genes that block antiviral defense mechanisms, has led to improved OV replication and tumor cytotoxicity (reviewed in ref. 36). When pharmacologic agents are used, the interference of antiviral responses can be applied in a transient fashion usually right before or at the time of OV administration. This should lead to an initial burst of OV replication leading to tumor cell lysis. As the pharmacologic effects against host innate immunity wane, a large debris field of OVs and tumor antigens could be more promptly recognized by the antiviral host response, leading to a secondary long-term vaccination effect responsible for effective tumor immunity (Fig. 1). However, quantification of responses to OV therapy is a sorely needed area of investigation. For instance, the number of OV-replicative rounds, the tumor cell-OV burst size, the number of OV-replicative tumor foci, and the temporal kinetics of innate response suppression that are needed for an efficient lytic and vaccination effect are still undetermined. In fact, current applications of innate immunity modulation with OV administration remain to be determined in an empirical manner.


'Secret weapon' of retroviruses that cause cancer

Oncogenic retroviruses are a particular family of viruses that can cause some types of cancer. Thierry Heidmann and his colleagues in the CNRS-Institut Gustave Roussy-Université Paris Sud 11 "Rétrovirus endogènes et éléments rétroïdes des eucaryotes supérieurs" Laboratory have studied these viruses. They have identified a "virulence factor" that inhibits the host immune response and allows the virus to spread throughout the body. This factor is a sequence of amino acids that is located in the envelope protein of the virus.

These scientists have also shown that once mutated to lose its immunosuppressive capability, this envelope protein could serve as a basis for the development of vaccines.

These findings have been published online in the Proceedings of the National Academy of Sciences USA.

Retroviruses are viruses whose genome is made up of RNA. These viruses are unique in possessing an enzyme that enables synthesis from this RNA of a DNA molecule capable of integrating into the DNA of a host cell. The retrovirus then utilizes the cell machinery to replicate. HIV is one of the best-known retroviruses. Oncogenic retroviruses (or oncoretroviruses) are cancer-causing viruses. Numerous oncoretroviruses are associated with animal diseases. In humans, two retroviruses, called HTLV and XMRV, have been associated with a type of leukemia and with prostate cancer.

Researchers in the Rétrovirus Endogènes et Eléments Rétroïdes des Eucaryotes Supérieurs Laboratory (1), headed by Thierry Heidmann, CNRS Senior Researcher at Institut Gustave Roussy, have been working on the ability of retroviruses to propagate and persist in their hosts by escaping the immune system. They have studied the molecular basis of this process, and have shown that it is driven by the envelope protein of these viruses. First of all, this protein has an essential "mechanical" role, as it induces the fusion of viral particles with the target cell membrane, thus allowing them to penetrate into the cell. Using a mouse model of infection with a murine leukemia virus, the researchers showed that this envelope protein also has a second role that is equally essential to viral propagation in the body: it is immunosuppressive, or in other words it inhibits the host immune response in a radical manner, affecting both the "innate" and "adaptive" immune responses.

The researchers succeeded in locating the domain responsible for this property within the amino acid sequence of the envelope protein. This domain, an authentic virulence factor, is a crucial element in the arsenal that enables retroviruses to invade their host and produce their pathogenic effect. It thus becomes a target of choice for the design of novel antiretroviral therapeutic strategies, including vaccines. The results obtained by these scientists mean it will be possible to follow this path.

hey were able to introduce targeted point mutations into the envelope protein that could suppress its ability to inhibit the immune system which, as expected, reacted much more effectively than with the non-mutated protein, producing a high level of antibodies and inducing antiviral cellular immunity. By working on this mutated protein, it should be possible to develop vaccines for the future. Indeed, after the mouse model, the researchers were able to show that the HTLV and XMRV retroviruses associated with human diseases were both endowed with an immunosuppressive domain in their envelope protein.


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