Co-infections with additional oncoviruses in HPV+ individuals: status, function and potential clinical implications

Abstract

Oncovirus infections account for an estimated 12% to 20% of human cancers worldwide. High-risk human papillomavirus (HPV) infection is the etiological agent of some malignancies such as cervical, oropharyngeal, anal, penile, vaginal, and vulvar cancers. However, HPV infection is not the only cause of these cancers or may not be sufficient to initiate cancer development. Actually, certain other risk factors including additional oncoviruses co-infections have been reported to increase the risk of patients exposed to HPV for developing different HPV-related cancers. In the current review, we summarize recent findings about co-infections with different oncoviruses in HPV+ patients from both clinical and mechanistic studies. We believe such efforts may lead to an interesting direction for improving our understanding and developing new treatments for virus-induced cancers.

Introduction

Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide (1). HPVs represent a family of non-enveloped, icosahedral, circular, double-stranded DNA viruses (2). The HPV genome contains approximately 8 open-reading frames (ORFs) encoding early (E) and late (L) proteins, as well as a largely non-coding long control region (LCR). The early proteins have regulatory functions in the infected epithelial cell, whereas L1 and L2 are structural proteins that form the viral capsid. The LCR contains cis-acting regulatory sequences that control viral replication, transcription, and post-transcriptional regulation (2). HPV infection is a well-established cause of cervical oropharyngeal, anal, penile, vaginal, and vulvar cancers, and of other associated diseases, including cutaneous and anogenital warts (3). Certain other risk factors such as smoking, may influence which women exposed to HPV are more likely to develop cervical cancer (4). In addition, some co-infected pathogens, such as human immunodeficiency virus (HIV) and chlamydia, have been reported to increase the risk of women exposed to HPV for developing cervical cancer (5, 6).

An oncovirus or oncogenic virus is a virus that can cause cancer. This term originated from studies of acutely transforming retroviruses in the 1950–60s (7), before it was known that both DNA and RNA viruses are capable of causing human cancers. Approximately 12% to 20% of human cancers worldwide are caused by oncovirus infection, with most cases occurring in the developing world (8, 9). Besides HPV mentioned above, other major human oncoviruses include hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), and human T cell lymphotropic virus-1 (HTLV-1). In this review, we summarize recent findings about co-infections of these oncoviruses in HPV+ patients from both clinical and mechanistic studies, which represents an interesting direction to improve our understanding and managing virus-induced cancers. We also include human cytomegalovirus (HCMV) here because its infection has been implicated in malignancies derived from breast, colorectal muscle, brain, and others (10).

HBV/HCV and HPV

HBV and HCV infections account for a substantial proportion of liver diseases worldwide. Both viruses can induce chronic liver disease, such as cirrhosis, and are notably associated with hepatocellular carcinoma (HCC). Because these two hepatotropic viruses share same modes of transmission, co-infection with these two viruses is not uncommon, especially in those high prevalence areas or among people at high risk for parenteral infection. Patients with dual HBV and HCV infection may have more severe liver disease, and are at an increased risk for progression to HCC (11). Liu et al performed a study to evaluate microorganism co-infections and associated risk factors in HPV-infected women in Northern China (12). In this study, cervical samples of 4290 enrolled female patients were collected to detect HPV, bacterial and yeast infections. Serum samples collected were analyzed for the presence of serological markers for HIV, HBV and others. Their statistical analyses revealed a significant association between HPV and detection of anti-HBV antibodies in these patients. However, it should be noticed that China has a high prevalence of HBV in general population, which definitely increasing the incidence rate of such co-infections. Another prospective study was conducted that enrolled 103 individuals from Brazil, among them, 48 participants tested positive for HBV and 55 tested positive for HCV; 2 had dual infection with HBV and HCV (13). Moreover, co-infection with HPV was detected among 20 participants, indicating that a high prevalence of co-infection with HPV was detected among Brazilian individuals with HBV and/or HCV. One interesting study evaluated the effects of HBV or HCV infection on the risk of cervical cancer, which enrolled 838 cervical cancer cases and 838 benign disease controls (14). They found that HBV infection was associated with cervical cancer in patients with age younger than 50 years (adjusted odds ratio = 2.1; 95% CI: 1.0–4.4), and individuals with both HBsAg and HPV positive had an increased risk of cervical cancer when compared with the benign controls (adjusted odds ratio = 67.1; 95% CI: 23.4–192.7).

When attempting to characterize the potential synergism between HPV and HBV/HCV in cancer development, we can note some commonalities in the molecular mechanisms (Figure 1) and integration patterns (Figure 2) in these viruses infected cells. Specifically, both HPV16/18 and HBV can target protein tyrosine phosphatase non-receptor type 3 (PTPN3) via its PSD-95/Dlg/ZO-1 (PDZ) domain (15). This phosphatase is known to have both oncogenic and tumor suppressor activity (16). Jing et al found that the PDZ-binding motifs (PBMs) of HPV E6 protein interacted with PTPN3 and caused its degradation by the proteasome, which also requires a cellular HECT domain ubiquitin ligase termed E6AP (17). This event could result in reduced cellular requirements for growth factors, leading to malignancy. Genera et al found that HBV-encoded HBc protein disrupted the interaction between PTPN3 and the polypyrimidine tract-binding protein (PTB, a PBM-containing partner of PTPN3) by competition, which could lead to more exportation of viral RNAs and promote virus production (18). In another interesting study, Ferber et al explored potential similarities in integration by HPV and HBV, specifically with regards to the human telomerase reverse transcriptase (hTERT) gene (19). They identified two HBV and three HPV integrations into the hTERT gene, in which four integrations occurred within the hTERT promoter and upstream region and the fifth integration occurred in intron 3 of the hTERT gene. None of the integrations altered the hTERT coding sequence, instead, all resulted in juxtaposition of viral enhancers near hTERT, potentially leading to hTERT activation (19). Therefore, these results suggest that the genes at the sites of viral integration may play important roles in virus-induced carcinogenesis.

Figure 1. Schematic of potential commonalities in the molecular mechanisms between HPV and HBV in tumorigenesis.

HPV E6 protein interacted with protein tyrosine phosphatase non-receptor type 3 (PTPN3) and caused its degradation by the proteasome, which also requires a cellular HECT domain ubiquitin ligase termed E6AP. This event could result in reduced cellular requirements for growth factors, leading to malignancy. HBV-encoded HBc protein disrupted the interaction between PTPN3 and the polypyrimidine tract-binding protein (PTB, a PBM-containing partner of PTPN3) by competition, which could lead to more exportation of viral RNAs and promote virus production.

Figure 2. Integration of HPV or HBV into the hTERT locus in cervical cancers (CCs) and hepatocellular carcinoma (HCCs).

Viral integrations into the CC tumors MC11, HK1, and HK2, the HCC tumor T89, and the HCC cell line SNU449 were shown. The nucleotide positions of the hTERT, HPV18, HPV16, and HBV sequences were in relation to accession numbers AY007685 (hTERT), X05015 (HPV18), K02718 (HPV16), AF068756 (HBV for T89), AF330110 (HBV for SNU449).

EBV and HPV

EBV is part of the human herpes virus family (gamma subfamily), DNA virus associated with a wide range of clinical manifestations. In particular, EBV is thought to be a common causative agent of autoimmune diseases in cases of chronicity/recurrency (20). Epithelial EBV infections have been linked to systemic lupus erythematosus and Sjögren’s syndrome, and EBV infection of B cells have been linked to multiple sclerosis and other diseases. Alongside this, EBV is also known for its oncogenicity, particularly leading to lymphoma and carcinoma (Burkitt’s lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma) (21). When investigating co-infections of EBV with HPV, it has been suggested that these viruses may interact to promote cancer development; both clinical and mechanistic data have supported this proposition. Here, we focus on the roles of their co-infection in malignancies traditionally associated with HPV, notably cervical and oropharyngeal cancers.

Many groups investigating the relationship between HPV and EBV with respect to cervical cancer progression found associations between the co-infection and higher lesion incidence and/or grade. Cameron et al studied 236 HIV+ American women over five years to compare the rates of squamous intraepithelial lesion (SIL) in the co-presence of HPV with a variety of human herpes viruses (22). They reported a trend that having high-risk HPV (hrHPV) and EBV co-infection increased the prevalence of SIL (including atypical squamous cells of undetermined significance, or ASCUS detection) when compared to hrHPV+/EBV− women (71.4% vs 47.7%). This group then looked at a new cohort of 295 HIV+ women, and they found that hrHPV infection alone did not significantly correspond to SIL incidence, but dual hrHPV/EBV positivity once again showed high association with SIL (~4 folds increase from hrHPV+ alone). Feng et al studied HIV+ Chinese women and found that women who were HPV/EBV positive had a higher incidence of high-grade cervical intraepithelial neoplasia (23). It should be noted that HIV infection may increase the incidence of HPV/EBV co-infection and HPV-related malignancies due to compromised cellular immunity. In a study of Iranian women, Joharinia et al reported similar trends. While they didn’t find statistically significant differences in their cervical lesion samples, they noted that EBV was mostly present in high-grade squamous intraepithelial lesions. They also found that all EBV positive samples harbored co-infections with hrHPV types 16, 18 and/or 31 (24).

These results, especially when combined, suggest that EBV may be an important cofactor in cervical cancer progression due to its associations with higher incidence of cervical intraepithelial neoplasias. Many groups have investigated the mechanisms through which EBV may influence HPV-associated oncogenesis. One group investigated the effects that both HPV and EBV infection may have on hypermethylation of the host genome. It was demonstrated that hypermethylation occurred via viral proteins interacting with the host genome to regulate DNA methyltransferase 1 (DNMT1), which can subsequently promote oncogenesis via hypermethylation (25). McCormick et al summarize current knowledge that HPV E7 protein inactivates the Rb1 tumor suppressor gene and downregulates E-cadherin via upregulation of DNMT1, while EBV has also been found to overexpress DNMT1 and subsequently lead to hypermethylation of CDH1 and decreased E-cadherin. In their work, they found that increased methylation of Rb1 and CDH1 corresponded to higher-grade cervical lesions. As for EBV’s contribution, there was not a significant association between cancer progression and EBV detection alone, but co-infection did significantly correspond to higher cancer rates. This suggests that although HPV is the main viral factor in cervical lesion progression, EBV’s co-presence with HPV is likely influencing oncogenesis and should continue to be investigated. Blanco et al investigated the role of BARF1, a lytic gene present in EBV-associated epithelial cancers (26). Although they didn’t find significant interdependence between BARF1 expression and cervical lesion grade, direct transfection of HPV16+ SiHa and CaSki cell lines with BARF1 vector suggested an oncogenic effect. In fact, they found that BARF1 increased proliferation and migration of these cells. They also assessed for BARF1-induced epithelial-mesenchymal transition (EMT), and found significant increase ZEB1 and insignificant but measured decrease of E-cadherin, suggesting potential influence by BARF1 in morphological changes and disease progression in the presence of high-risk HPV.

In the case of HPV-associated oropharyngeal cancer, there is less work focused on population statistics and mechanistic studies. With a small sample size of 63 men, Dickey et al presented data suggesting that EBV co-infection may promote the persistence of HPV16/18 infection in the oral cavity (27). Based on these results, the authors suggest that EBV DNA detected in the oral cavity may be a useful biomarker which may indicate reduced of immunity to respond to oral infections. Strycharz-Dudziak et al performed a study with 128 patients investigating the effects the co-infection can have on oxidative stress management as well as its influence on overall malignant transformation (28). While they recognized a small size as a limitation of their study, they reported decreased activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in the cases of HPV/EBV dual positivity when compared to single infections. They also found decreased antioxidant activity in the cases of poorly differentiated tumors, as well as greater tumor dimensions and greater lymph node involvement. They suggested co-infection as a possible means for evading antioxidant protective endeavors. However, further studies are needed to clarify the influence of interplay between EBV, HPV and oxidative stress on malignant transformation of upper aerodigestive tract epithelial cells.

KSHV and HPV

KSHV represents a principal causative agent of several human cancers arising especially in those immunocompromised patients, including Kaposi’s Sarcoma (KS), Primary effusion lymphoma (PEL) and Multicentric Castleman’s disease (MCD) (29). Currently, there are limited data reporting HPV and KSHV coinfection in clinical cases. One study about KSHV infection in sex workers and women from the general population in Spain reported that KSHV DNA was detected in 2% of the cervical samples of the prostitutes and in 1% of the cervical samples of women in the general population (30). Moreover, the authors found that KSHV was more prevalent among HPV DNA-positive women (odds ratio = 2.5). Another study reported oral coinfection of HPV and KSHV among HIV-infected individuals in Italy, especially men who have sex with men (MSM) (31).

We recently reported that KSHV can established latent infection in a variety of HPV+ cervical cancer cell lines such as HeLa, SiHa and CaSki (32, 33). We found that KSHV in latently infected cervical cancer cells possess normal replicative potential, such that the virus can be induced into lytic phases by exogenous stimulus and can produce new infectious particles (32). Interestingly, our data indicated that KSHV infection significantly reduced both E6 and E7 expression from HPV16+ SiHa cells in vitro (32). By using a cervical cancer xenograft model, we also confirmed these results in vivo (33). Furthermore, we found that LANA (Latency associated nuclear antigen) and vFLIP (viral FLICE inhibitory protein), two major KSHV-encoded latent proteins, were responsible for the downregulation of E6 and E7 expression from SiHa cells (32). In contrast, another group found that one of KSHV-encoded lytic protein, RTA (Replication and transcription activator), can bind to various HPV16 genomic regions and induce a significant upregulation of E7 transcription (34). We speculate that KSHV latent and lytic proteins may have distinct regulation of HPV oncogenic proteins expression in cervical cancer cells. Furthermore, it will be interesting to understand whether these events are due to a competition during virus co-infection rather than regulation.

We found that KSHV infection significantly increased macrophage migration inhibitory factor (MIF) secretion from HPV+ cancer cell lines such as SiHa and CaSki (5–8 folds increasing) (33). MIF enters target cells by binding to its cellular receptors such as CXCR2, CXCR4 or CD74 (35, 36). One previous study reported the overexpression of MIF in invasive cervical cancer samples when compared to cervical dysplasia samples (37). Another study found that MIF and CD74 expression was significantly higher in CIN or CSCC than in the normal samples (38). Therefore, our findings suggest that KSHV co-infection may represent one possible novel mechanism of upregulating MIF, its receptors and downstream signaling (e.g., ERK1/2, PI3K, HIF1α) in cervical cancer cells (Figure 3). Therefore, MIF may be an attractive therapeutic target against cervical cancer.

Figure 3. Schematic of potential impacts of KSHV co-infection on HPV+ cervical cancer pathogenesis.

LANA: Latency associated nuclear antigen; vFLIP: viral FLICE inhibitory protein; MIF: Macrophage migration inhibitory factor; CXCR2: C-X-C motif chemokine receptor 2; CXCL1: Chemokine (C-X-C motif) ligand 1; CCL5: Chemokine (C-C motif) ligand 5; IL-6, Interleukin 6.

HTLV1 and HPV

HTLV1 is a retrovirus that causes lifelong T-cell infection in humans, impacting the host immune response. This virus causes a range of clinical manifestations, especially adult T-cell leukemia (ATL) (39). Studies concerning coinfection with HPV and HTLV1 are limited. One study from Brazil reported that the prevalence of HPV infection was higher among HTLV1 infected than uninfected women attending a gynecology clinic (44% vs. 22.5%, p = 0.03) (40). In HTLV1 infected women from the Peruvian Amazon, the prevalence of HPV infection was also higher than in HTLV-1 negative individuals (43.6% vs 29.3%, prevalence ratio = 2.1; 95% CI: 1.53–2.87). The prevalence of high-risk HPV infection was also higher (32.3% vs 22.4%, prevalence ratio = 1.93; 95% CI: 1.04–3.59) than those not infected by HTLV1, after adjusting for confounding variables (41). The HTLV1 is endemic in some countries in Sub-Saharan Africa. One study reported an unexpectedly high prevalence of HTLV1 DNA in HIV-positive women in Kenya (42). They reported that 22/113 (19.5%) of liquid based cytology (LBC) cervical smears from HIV-positive patients were positive for HTLV1 compared to 4/111 (3.6%) of those from HIV-negative women (p = 0.0002; odds ratio = 6.42; 95% CI: 2.07–26.56).

Both HTLV and HPV have high-oncogenic and low-oncogenic subtypes, and such subtype-specific oncogenesis is associated with the PDZ-domain binding motif (PBM) in their transforming proteins. HTLV-1 encodes Tax1 with PBM as a transforming protein. Aoyagi et al constructed Tax1 mutants with PBM from HPV-16 E6 and they showed that it was fully active in the transformation of a mouse T-cell line from interleukin-2-dependent growth into independent growth (43). They also found that Tax1 and Tax1 PBM mutant interacted with tumor suppressors Dlg1 and Scribble with PDZ-domains. Unlike E6, Tax1 PBM mutant as well as Tax1 did not or minimally induced the degradations of Dlg1 and Scribble. Rather, they induced their subcellular translocation from the detergent-soluble fraction into the insoluble fraction, suggesting that the inactivation mechanism of these tumor suppressor proteins is distinct. Their results suggest that PBMs of high-risk oncoviruses have a common function required for these oncogenic viruses to transform cells. Interestingly, hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was found to interact with both HPV-16 E7 and HTLV1 Tax1 protein, which are required for cellular transformation induced by these oncogenic viruses (44, 45).

HCMV and HPV

HCMV is a herpesvirus that is pervasive in the human population with the global seroprevalence of 60–90% (46). HCMV infection tends to be asymptomatic in people with a competent immune response, but the primary HCMV infection can cause up to 7% of mononucleosis syndrome which is indistinguishable from that caused by EBV (47). HCMV can spread through many avenues– sexual transmission, blood transfusions, organ transplants, vaginal birth, and other contact with bodily fluids (48). Following initial infection, and usually after a period of latency, this virus can later cause more severe or life-threatening issues, most notably various cancers (breast, brain, and colorectal) (10, 46). Paradowska et al investigated prevalence of HCMV and HPV in fallopian tubes specimens and tumor tissues from epithelial ovarian carcinoma (EOC) patients (49). The presence of HCMV and HPV DNA was detected in 70% and 74% cancerous ovarian tissues, respectively, and was significantly higher in EOC than in benign tumor cases (p ≤ 0.01). HCMV or HPV infection was also observed in the fallopian tube samples. HPV16 DNA was specifically denoted as being more prevalent in EOC cases when compared to the control, although no statistical significance due to small sample size (p = 0.039). Furthermore, they found that 17 of 27 patient samples (63.0%) had both HCMV and HPV infection. If considering co-infection of HCMV and HPV16, they found that it was significantly higher in EOC cases when compared to the control (p = 0.004). Finally, the researchers concluded that the odds of co-infection in EOC cases was 24 times higher than that in benign tumor controls (odds ratio = 24.39; 95% CI: 1.28–466.30) (49).

Cao et al. investigated host immune response in virus-associated cancers, and reported the upregulation of several immune checkpoint markers, such as PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4–1BB in HCMV positive tumors in comparison to virus negative tumors (50). In a few HPV positive head and neck squamous cell carcinoma (HNSCC) samples, they found that the HPV genome integrated in immune checkpoint genes PD-L1 or PD-L2, driving elevated expression in the corresponding genes. In fact, it has been reported that some HPV proteins (e.g., E5 and E7) are able to upregulate PD-L1 expression (51). Therefore, HPV involvement, whether via integration or not, shows direct interaction with PD-L1 pathways. Although Cao et al do not address virus co-infection in their work, their findings clearly support mechanistic commonalities between HPV and HCMV in regulation of PD-L1/PD-L2 expression (50). In an interesting review article, Marongiu and Allgayer highlighted similarities of the mechanisms of oncogenic DNA viruses, and their relationship to colorectal cancer (CRC) oncogenesis (52). They found that there were striking common features of HCMV, HPV, and other oncoviruses in that they were able to promote oncogenesis by disrupting the cell cycle by targeting p53, pRb, and p21, interfering with epithelial-mesenchymal transition (EMT)-/EGFR-associated pathways, and targeting the Wnt/β-catenin-driven pathways. Therefore, the oncoviruses co-infections may cause an additive effect on tumor initiation or progression in some CRC patients.

Conclusion and prospective

An increasing amount of evidence has indicated the potential roles of other oncoviruses co-infection in HPV-related malignancies development. However, most of these data come from clinical or retrospective studies, and mechanistic studies especially those using co-infection cell culture systems and/or co-infection animal models are lacking. In terms of co-infection, it does not necessarily mean that two oncoviruses must infect the same cells. In fact, virus-infected cells can release cytokines, chemokines, other factors, or even exosomes and microvesicles to affect neighboring cells and microenvironment. Interestingly, previous studies have suggested that some pathogens co-infections may contribute to the process of HPV carcinogenesis without the need for continued presence (the hit-and-run theory) (53, 54). As mentioned above, one should be aware of potential synergism of oncoviruses co-infections in cancer development, which may increase the risks of HPV+ patients with other oncoviruses co-infections for developing HPV-related and other cancers. One of major challenges is that currently there is a lack of good in vivo experimental models to directly study the co-infections of HPV and other oncoviruses. Therefore, whether they may have synergistic effects on tumor formation is very difficult to address. Actually, some oncoviruses such as KSHV do not even have their own good in vivo infection models. Also, virus-induced tumorigenesis may be a long-term process which is somehow difficult to address in the experimental models in vivo.

Data availability

All the data shown in this paper are available from the corresponding authors upon reasonable request.