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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 15  |  Issue : 3  |  Page : 155-163

MMTV-like Env sequences from human breast cancer patients cannot yet be considered as a separate species


1 Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
2 Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University; Zayed Center for Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates

Date of Submission15-Apr-2022
Date of Decision26-May-2022
Date of Acceptance01-Jun-2022
Date of Web Publication21-Sep-2022

Correspondence Address:
Farah Mustafa
Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, Tawam Hospital Complex, P.O. Box 15551, Al Ain
United Arab Emirates
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/hmj.hmj_35_22

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  Abstract 


Background: Mouse mammary tumour virus (MMTV), a betaretrovirus, causes breast cancer in mice. Since its discovery, scores of studies have reported the detection of MMTV-like antigens and sequences primarily in human breast cancer, but not normal tissues. The presence of these sequences in humans has been hypothesised to be possibly due to zoonosis of MMTV into humans, named human mammary tumour virus (HMTV). However, many groups have not been able to repeat these findings, making these observations controversial. Over the years, an increasing number of HMTV env gene sequences from human breast cancer patients worldwide have been deposited in GenBank and other repositories. Aims and Objectives: The aim of this study was to use the current bioinformatic tools to analyse these highly homologous sequences to determine if any signature sequences could be associated specifically with HMTV. Materials and Methods: We first built an MMTV env gene consensus sequence (MMTV_CON) from the 41 MMTV sequences available in the database that was used to align the reported HMTV sequences (n = 333). Results: As expected, the MMTV envs showed 4-5% genetic variation within the mouse isolates. Alignment of MMTV_CON with those from HMTV revealed ten nucleotide variations that were like those observed within MMTV env, showing that the two viral strains could not be distinguished. Conclusion: Thus, we conclude that despite extensive data, inadequate env coverage, conservation of MMTV and HMTV envs and limitations in HMTV study design suggest that HMTV cannot be considered a separate species until the availability of more data covering full-length env or HMTV genome.

Keywords: Bioinformatics., env gene, human mammary tumour virus, mouse mammary tumour virus, retrovirus, zoonosis


How to cite this article:
Ahmad W, Khader TA, Panicker NG, Akhlaq S, Baby J, Gull B, Mustafa F. MMTV-like Env sequences from human breast cancer patients cannot yet be considered as a separate species. Hamdan Med J 2022;15:155-63

How to cite this URL:
Ahmad W, Khader TA, Panicker NG, Akhlaq S, Baby J, Gull B, Mustafa F. MMTV-like Env sequences from human breast cancer patients cannot yet be considered as a separate species. Hamdan Med J [serial online] 2022 [cited 2022 Oct 7];15:155-63. Available from: http://www.hamdanjournal.org/text.asp?2022/15/3/155/356433




  Introduction Top


Breast cancer is the most common form of cancer in women. Only 5-10% of breast cancer cases are familial due to mutations in BRCA1 and BRCA2 genes, the remaining being sporadic in nature.[1] Many risk factors are associated with breast cancer initiation, development and survival. These include age, breastfeeding, menopause, hormonal and immunological record of the patient, as well as environmental factors such as diet, smoking, life style, exposure to chemicals, pesticides and fertilisers.[2],[3] It is estimated that 16% of all cancer cases in humans are attributed to infectious agents;[4] therefore, there is a chance that microorganisms may be involved in the development and progression of breast cancer in humans.[5]

The mouse mammary tumour virus (MMTV) is one such viral pathogen that causes breast cancer in mice and increasing evidence suggests that it may be involved in human breast cancer as well. MMTV is a classic betaretrovirus that uses insertional mutagenesis to induce breast cancer.[6] It was first reported as an 'extrachromosomal' milk-borne agent with the ability to induce breast cancer in 1936.[7] Since its identification as a retrovirus,[8] there has been an intensive search to look for a human counterpart of MMTV, human mammary tumour virus (HMTV), both at the antigenic and sequence levels.[5],[8],[9] The first MMTV-like env sequences were reported in human breast cancer tissues which were essentially absent in normal tissues in 1995 using polymerase chain reaction (PCR).[10] Since then, there has been a slew of studies reporting such sequences primarily in breast cancer, but not in normal breast tissue from all over the world.[9],[11],[12] However, the detection of MMTV sequences in human samples is not consistent and many studies have reported the absence of HMTV (MMTV-like) sequences or lack of their association with human breast cancer samples.[11],[13] MMTV uses its envelope (env) gene to interact with the mouse transferrin receptor I (mTfR1) for cellular entry in mice; however, the human transferrin receptor I does not support this entry.[11],[14] This is despite the fact that the mouse and human transferrin receptors have 86% homology at the amino acid levels and the MMTV and HMTV envelop (env) gene sequences are >95% homologous.[11],[14] These and other observations have raised questions about the presence of MMTV sequences in human tissues since primarily PCR was used for their detection, suggesting the potential of MMTV contamination as a plausible explanation.

As can be seen, MMTV as a possible cause of breast cancer in humans is controversial. With the increasing number of MMTV-like sequences being reported from all over the world and their sequences deposited in various databases, the purpose of this study was to conduct a thorough bioinformatics analysis of these sequences compared to those from MMTV to determine whether any potential sequence signatures could be observed that were not apparent when only limited HMTV sequences information was available. We focused on the env gene since it is the most interrogated region by most groups using PCR. Furthermore, it is the gene primarily involved in viral tropism, determining its host range and entry into cells. Therefore, we first retrieved essentially most of the reported HMTV sequences from the nucleotide repositories and publications that could be located and aligned them with the MMTV env consensus sequence to find any potential differences and/or variations that could distinguish the two genes. Based on the sequences available, our analysis confirms that HMTV and MMTV env sequences are indistinguishable at the sequence level in the region analysed; however, lack of full gene coverage remains a major caveat of this analysis, and until more sequence data becomes available, it remains inconclusive whether HMTV can be called a separate strain of MMTV.

Methods

Sequence retrieval

MMTV and HMTV genome sequences were retrieved from NCBI Nucleotide (https://www.ncbi.nlm.nih.gov/nuccore/) and EMBL-EBI ENA (https://www.ebi.ac.uk/genomes/) databases. Furthermore, published data were thoroughly searched using PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Google Scholar (https://scholar.google.com/). Some HMTV sequences were not available in databases and were directly copied from the published manuscripts or requested from authors. We are thankful to Dr. Vitiello[15] for sharing their unpublished HMTV sequences. All sequences containing regions other than env were cropped manually to assist with sequence alignment.

Sequences alignments

The MMTV env consensus sequence (MMTV_CON) was generated by submitting available MMTV sequences to a multiple sequence alignment application MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) followed by submission of the generated FASTA file to the NCBI Multiple Sequence Alignment Viewer (https://www.ncbi.nlm.nih.gov/projects/msaviewer/). The same file was used to create the sequence logos using WebLogo 3 (http://weblogo.threeplusone.com/create.cgi) using default settings.

Phylogenetic analysis

MUSCLE was used to check the homology of MMTV env consensus sequence with available MMTV genomes and FigTree v1.44 (http://tree.bio.ed.ac.uk/software/figtree/) was used for viewing the phylogenetic tree. SimPlot v3.5.1 (https://sray.med.som.jhmi.edu/SCRoftware/SimPlot/)[16] was used to view differences among three widely used MMTV genomes compared to MMTV_CON.

Human mammary tumour virus sequence alignment with the mouse mammary tumour virus env gene consensus sequence

Due to the limitation of commonly used alignment programs, the HMTV sequences were submitted to the online Galaxy database (https://usegalaxy. org/).[17] A fast and reliable alignment tool Bowtie2 at default settings was used to align the HMTV sequences with MMTV_CON. The BAM output file was viewed using IGV v2.8.2 (https://software.broadinstitute. org/software/igv/bam) and the sequence logo was generated by WebLogo 3.[18] The BAM file was further converted to FASTA format using ConvertBAM option in Galaxy server.

Alignment of the mouse mammary tumour virus env gene consensus sequence with HERV-K and human genome

The EMBOSS Water Pairwise Sequence Alignment web application (https://www.ebi.ac.uk/Tools/psa/emboss_water/) was used to find any similarities between MMTV env and HERV-K (DQ112122.1). For alignment with the human genome assembly GRCh37 (hg19), the Galaxy application Bowtie2 was used at default settings.


  Results Top


The study started with data collection of MMTV and HMTV sequences from the available databases, published studies and personal communications. A total of 7 MMTV full-length genomes, 34 MMTV env genes and 333 HMTV env sequences were successfully collected. No HMTV full-length genomic data could be located in the databases.

To compare the MMTV and HMTV env sequences, we first generated an MMTV env consensus sequence, MMTV-CON, which was aligned with 333 HMTV sequences collected from the databases, literature as well as investigators. [Figure 1]a shows the MMTV full-length genome from the C3H strain (accession no AF228552.1), while [Figure 1]b shows its env gene. The various crucial elements within the 2067 bp gene are highlighted, including the reported splice sites, internal env promoters and env transcriptional activator.[19],[20],[21] [Figure 1]c shows the protein products of MMTV env which encodes a 98 amino acid signal peptide (SP; p14) and the 590 amino acid gp73 glycoprotein. gp73 is further cleaved by host furin protease into the mature envelope proteins gp52 and gp36.[22] gp52 is the surface (SU) domain of Env that interacts with the viral host receptor, the mTfR1, using its receptor binding domain (RBD), while gp36 is the transmembrane™ domain responsible for the fusion of the viral envelope with the host endosomal membrane, allowing entry of the viral capsid carrying the dimerized genomic RNAs into the cells. Finally, [Figure 1]d shows the location of the HMTV reads that were collected from various sources with respect to the MMTV env gene.
Figure 1: Schematic representation of MMTV genome and env. (a) MMTV genome (C3H strain). (b) MMTV env gene with marked cis-acting elements. (c) Illustration of Env peptides/proteins. (d) HMTV sequence alignment along the C3H env. HMTV: Human mammary tumour virus, RBD, receptor binding domain; HBD, heparin-binding domain

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Mouse mammary tumour virus env sequences have <5% variation among isolates

We retrieved 41 MMTV env sequences from the online databases that were aligned using MUSCLE. The generated FASTA file was submitted to the NCBI Multiple Sequence Alignment Viewer to find any dissimilarities among them and to construct a consensus sequence. The length of most of the MMTV env sequences was complete and similar (n = 31), whereas some of them covered either half (n = 5) or partial gene (n = 5) [Figure 2]a. We found a total of 96 single nucleotide polymorphisms (SNPs) among all MMTV env sequences when compared with the consensus sequence, MMTV_CON [Figure 2]b. This showed a ~4.5% variance among all sequences. Most of the variations/polymorphisms within the MMTV env genes were a transition between A ↔ G or C ↔ T. Transverse mutations were observed at nucleotide positions 10, 63,119, 140, 402, 858, 888, 1098, 1675, 1701, 1725, 1887, 1905 and 1911, whereas both transition and transverse mutations were observed at nt positions 146, 177, 378, 504, 633, 844, 966, 1377, 1659 and 1675. However, a majority of the MMTV env genes showed sequences similar to the consensus. These similarities and differences have been represented as sequence logos [Figure 2]c where nucleotides (nt) at the same positions were scaled according to their frequency. The conserved residues have more height than those with differences, whereas the thickness of the residues represents the number of sequences containing such residues.
Figure 2: MMTV env variation analysis. (a) Alignment of MMTV envs to generate the consensus MMTV_CON. The red vertical lines show sequence variations. (b) Sequence logos of aligned MMTV sequences. (c) Analysis of the 96 polymorphisms observed in MMTV envs

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Mouse mammary tumour virus env consensus sequence is closest to the mouse mammary tumour virus GR isolate

To check which known MMTV env isolate was closer to the consensus sequence, MMTV_CON, we performed phylogenetic analysis and created the SimPlots. MUSCLE was used to create phylogenetic tree data that was visualised using FigTree software. Phylogenetic analysis along with pair-wise alignment analysis showed a higher similarity of MMTV_CON (97.5%) with MMTV isolate GR (accession no: XO1811.1), whereas the MMTV env isolate AK033592.1 showed the least similarity (96.3%) [Figure 3]a. SimPlot was used to identify differences between MMTV_CON with the three most commonly studied MMTV strains, including GR (X01844.1), BR6 (M15122.1) and C3H strain (AF228552.1). SimPlot analysis revealed that the first ~800 nucleotides of MMTV_CON were closer to the C3H strain with higher similarity (~100%), whereas the middle part (from ~ nt 801–1400) was similar to all three strains with some nucleotide changes (~98% similarity among all sequences). Interestingly, the latter part of the env gene (~ nt 1401–2067) of MMTV_CON showed comparatively lower (~93%–98.5%) similarity among these three strains [Figure 3]b. These results reveal that our consensus sequence represented all MMTV strains.
Figure 3: Phylogenetic analysis of the MMTV env consensus (MMTV_CON). (a) Phylogenetic tree showing relationship of MMTV_CON with other MMTV strains. (b) SimPlot showing variations among GR (X01811.1), C3H (AF228552.1) and BR6 (M15122.1) compared to MMTV_CON. MMTV_CON: Mouse mammary tumour virus env gene consensus sequence

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Analysis of human mammary tumour virus envs

The first MMTV-like env gene sequence was submitted to the NCBI Nucleotide database in 1998.[23] Since then, many countries around the globe, especially researchers from Australia, USA and Mexico, have observed and submitted MMTV-like env sequences partially covering the gene. In this study, we aimed to collect as many available HMTV sequences as possible. We found 333 HMTV sequences that were either submitted to the NCBI or published in various manuscripts. [Table 1] and [Figure 4]a comprehensively describe the year, country and group reporting the HMTV sequences. Most of the sequences were published/submitted by Australian researchers (137 sequences, 10 studies), followed by the USA (54 sequences, 8 studies) and Mexico (51 sequences, 3 studies). Two studies from Italy reported nine sequences, while single studies from Brazil, China, Iran, Morocco, Myanmar, Pakistan, Saudi Arabia, Tunisia and UK reported 28, 1, 1, 24, 1, 1, 8, 10 and 3 HMTV sequences, respectively. Our analysis shows that Australia, USA and Mexico reported the most sequences. Furthermore, most of these sequences came from the same investigators from Australia and the USA.
Figure 4: Alignment of HMTV sequences with MMTV_CON and their characterisation. (a) Number of MMTV-like (HMTV) sequences (Seq) and studies (Std) reported from different countries across the world analysed in this study. Size of square is relative to the proportion of sequences/studies reported from the indicated country. (b) Alignment of 333 HMTV sequences with MMTV_CON. Gray lines = conserved regions; vertical red, purple or green lines/dots show polymorphisms within the HMTV sequences compared to MMTV_CON. Blue dots show insertions/deletions. MMTV_CON: Mouse mammary tumour virus env gene consensus sequence, HMTV: Human mammary tumour virus

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Table 1: Summary of the sequences included in this study

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Human mammary tumour virus envs show higher similarity with mouse mammary tumour virus env gene consensus sequence

To find any differential signatures between MMTV and HMTV sequences, we first aligned the HMTV sequences with MMTV_CON using MUSCLE. Visualisation of the results showed multiple gaps between the sequences, indicating that the program was not suitable for this purpose [Figure S1]. Next, we used Bowtie2, a highly sensitive and most reliable multiple sequence alignment tool used for short and long reads. Our results showed that out of 333 HMTV sequences, 317 (95%) completely aligned with the MMTV consensus sequence. Of these, most (310; 98%) sequences aligned within nt 7240–7910 (middle of the env gene, 630 nt long), three sequences covered nt 6750–7910 (1200 nt long), while three others aligned along the nt 7657–8568 region (900 nt long). There was a single sequence of 1100 nt length that aligned along the nt 6561–7654 region of the MMTV C3H strain [Figure 1] and [Figure 4]b. Interestingly, most of the studies used primers that covered the MMTV env middle region away from the RBD. There were only four sequences that aligned in the 5' region containing the RBD, and these showed high conservation for this region [Figure 1]d. [Table 1] summarises the studies reporting MMTV-like sequences from human breast cancer across the globe, while Supplementary Data S1 provides the E-values for 240 of these sequence alignments along with other statistics that could be retrieved from NCBI.

As described above, we found 16 HMTV sequence entries that remained unaligned using Bowtie2. These sixteen sequence entries were then individually aligned with the MMTV consensus sequence to determine why they could not align. Although we could find >90% homology to MMTV env, Bowtie2 rejected these sequences due to the presence of gaps in the alignment as well as some heterologous flanking regions that did not align with MMTV-CON, creating bigger gaps, as observed in the sequence logo [Supplementary Data S2 and Figure S1].[Additional file 1]

Overall, our results showed a higher similarity of HMTV sequences (~97.5%) with MMTV_CON over the aligned region. We found only ten polymorphisms across the HMTV env sequences analysed when compared to the MMTV consensus sequence [Figure 5]. There were a few insertions/deletions in the HMTV sequences appearing at the start or end of the sequences; however, these could be due to PCR errors. All of these were transition mutations where A ↔ G (n = 9) or C ↔ T (n = 1). Despite these differences, a majority of the HMTV sequences were like the consensus sequence. Thus, if the consensus had G, the majority of HMTV sequences also had G and the remaining had A and vice versa [Figure 5].
Figure 5: Sequence differences between HMTV env and MMTV_CON. (a) HMTV sequences showed ten transition variations when aligned with MMTV_CON. (b) Sequence logos of aligned HMTV sequences. Taller height equals sequence conservation, whereas thickness represents number of sequences containing such residues. MMTV_CON: Mouse mammary tumour virus env gene consensus sequence, HMTV: Human mammary tumour virus

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Human mammary tumour virus sequences do not match with HERV-K or the human genome

One of the confounding factors of whether MMTV-like sequences exist in humans has been the presence of human endogenous retroviruses (HERVs) that have sequence similarity to MMTV,[53] with the latest endogenous virus HERV-K having the most similarity.[54] Thus, the possibility exists that the MMTV-like sequences detected in humans are due to the cross-reactivity of the primers with HERV-K sequences. This possibility has been explored by many investigators that have tried to circumvent this problem by designing primers that do not bind to HERV-K sequences.[9],[10] To find any relationship between HERV-K and the HMTV sequences, we aligned the reported 310 HMTV sequences corresponding to the ~800–1200 bp region of MMTV env with the full-length HERV-K sequence (DQ112122.1) and the human genome assembly GRCh37 (hg19) using Bowtie2. However, none of the HMTV sequences aligned with HERV-K or the human genome assembly GRCh37 (hg19) (data not shown). Similar findings were observed when these sequences and MMTV_CON were aligned with HERV-K using MUSCLE (data not shown). These results confirm the earlier observations that HMTV and HERV-K sequences are different and distinct from each other.[14]


  Discussion Top


This study was undertaken to determine whether signature sequence differences between MMTV and HMTV could be discerned using the power of the latest bioinformatics tools owing to the increasing number of HMTV sequences being reported from all over the world. Our results reveal that despite the increase in data on HMTV sequences over the years and from varied geographical areas, no signature sequences could be identified that could distinguish HMTV from MMTV envs. The biggest reason behind this result is probably the fact that most investigators interrogated a similar ~700 bp region (~nt 7240–7908) of the 2067 bp env gene (~1/3rd of the gene) using already published primers to avoid cross-reactivity with HERV-K. This leaves the possibility wide open that if more sequence information can be obtained over larger portions of the env gene or other parts of the HMTV genome, a better assessment of its relationship to MMTV can be made.

This is especially true for the env gene since the region encompassed by the HMTV sequences reported in the database is within the SU domain of Env that interacts with the viral cellular receptor, mTfR1. Within SU, RBD is the region used to interact with mTfR1; therefore, sequence diversity within this region could provide important clues to its interaction with its human receptor as well, while sequence variation outside the RBD may reflect either a different step of viral entry or a different mode of entry. As mentioned earlier, the human TfR1 (hTfR1) does not support the successful entry of MMTV into human cells, even though it can bind MMTV efficiently.[55] Furthermore, the MMTV envelope-bound TfR1 complex is only internalised and trafficked to a low pH compartment in cells expressing mouse but not human TfR1. Thus, while hTfR1 supports cell–cell fusion, because the virus-receptor complex is not internalised, it is thought not to function as an entry receptor.[55] This makes analysis of the TM domain of the env gene especially important since it allows fusion of the viral capsid with the endosomal membranes necessary for successful infection, an area of the env gene that has not been interrogated in human cancer tissues.


  Conclusions, Study Limitations and Future Directions Top


Based on the analysis presented, we conclude that HMTV cannot be considered a separate species of MMTV until more data covering full-length env or HMTV genome become available. The lack of these data also makes it an important limitation of this study since 98% of the HMTV sequences identified from the databases covered the same small 700 bp region of env. Thus, while other parts of the MMTV genome have been identified in breast cancer samples, more work needs to be done to identify all parts of the viral genome by independent investigators. Furthermore, only one study claims to have cloned two whole HMTV proviral genomes with ~95% homology to MMTV from two breast cancer samples,[56] but this needs to be repeated by other investigators independently. Considering that so far, no one has been able to repeat this observation even after 20 years of intensive investigations, suggesting that this may have been due to contamination. Finally, the most conclusive data that can put an end to this controversy in our view are to identify bonafide MMTV integrations in the human genome of actual breast cancer samples, a feat that so far has eluded the scientific community.

Ethical clearance

No ethical approval was required for this study as per Arrive Guidelines 2.0 clause 14 as this study reports an in silico analysis of previously published sequence data and did not involve experiments on human or animal subjects. The human and mouse sequence data used was curated from published studies and public databases and ethical permission for these studies was obtained at the time when the respective study was conducted.

Financial support and sponsorship

This study and TAK (a PhD student) were supported by a grant to FM from the Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Sciences (MRG-421 2019-2020).

Conflicts of interest

There are no conflicts of interest.



 
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