Comprehensive Analysis of Codon Usage Bias in Human Papillomavirus Type 51

Human papillomavirus (HPV) belongs to the Papillomaviridae family, characterized by its doublestranded DNA structure, recognized for causing various diseases in humans, such as cervical cancer, genital warts, and other anogenital cancers (Muñoz et al. 2003; de Sanjose et al. 2010; Tan et al. 2022). HPV is categorized into high-risk and low-risk types based on the severity of its pathogenicity (Liu et al. 2021). The continued presence of high-risk human papillomavirus (HR-HPV) is strongly associated with the development and progression of cervical cancer (Zhao et al. 2009). HPV-51 falls under the high-risk category, indicating its potential to cause abnormal cell changes leading to cancer if untreated (de Sanjose et al. 2010). HPV-51 DNA has been identified in both low-grade and highgrade cervical lesions, indicating its involvement in the progression of cervical cancer (Smith et al. 2007).

Furthermore, HPV-51 has been implicated in various anogenital cancers and lesions, such as vulvar, vaginal, penile, and anal cancers (Muñoz et al. 2003). While HPV-16 and HPV-18 are commonly acknowledged as high-risk types, HPV-51 has been identified in a significant proportion of cervical cancer cases globally, emphasizing its relevance in HPV-related diseases. A study highlights that while HPV-51 is less common among high-risk types, it still accounts for a notable percentage of HPV-related cancers (Smith et al. 2007). The prevalence of HPV-51 varies by region, but it has been detected in cervical, anal, and oropharyngeal cancers. While HPV-51 may not be as widespread as some HPV types, its potential to cause cancer underscores its importance in HPV-related disease prevention and management (Smith et al. 2007).

Codon bias is the phenomenon wherein synonymous codons coding for the same amino acid are not uniformly utilized within an organism’s genome (Sharp and Li 1986). This phenomenon is prevalent across diverse organisms, including viruses (Sharp et al. 1988). Understanding codon bias offers insights into evolutionary dynamics, translational efficiency, and host adaptation strategies (Das et al. 2006). Several factors influence an organism’s codon usage pattern, such as the abundance of specific transfer RNAs (tRNAs) for each codon, mutation bias, and natural selection (Plotkin and Kudla 2011). High-frequency codons, commonly found in highly expressed genes, often align with abundant cellular tRNAs, promoting efficient translation. Conversely, low-frequency codons may lead to less efficient translation due to the limited availability of corresponding tRNAs, potentially affecting protein synthesis rates (Koonin and Novozhilov 2009). Viruses often adjust their codon usage to match that of their hosts, which improves viral replication and helps them avoid host immune responses (Hershberg and Petrov 2008; Chaney and Clark 2015). This adaptation underscores the importance of codon usage bias in understanding the evolutionary processes shaping genomes and optimizing heterologous gene expression for biotechnological applications (Quax et al. 2015). Codon usage can affect protein folding, stability, function, and the yield of recombinant proteins in expression systems. Consequently, codon optimization strategies are often employed to boost the expression of foreign genes in host organisms, especially in microbial systems used for biopharmaceutical production (Sharp and Devine 1989).

To date, there have been no reports on the codon preference of HPV-51. Given its clinical relevance and its association with various cancers, understanding its codon usage patterns could offer valuable insights into its evolutionary dynamics and interaction with host cellular machinery. This study aims to fill this gap by providing a comprehensive analysis of the codon usage bias of HPV-51.

HPV-51 belongs to the high-risk category of HPV, which is linked to multiple cancer types, such as cervical cancer. Although of clinical importance, scant attention has been paid to studying its codon usage bias. The research presents a comprehensive analysis of codon usage bias in HPV-51, shedding light on its evolutionary dynamics, host adaptation strategies, and potential translational efficiency within human cells. In our study, we thoroughly investigated codon usage patterns by analyzing coding sequences from 64 genomes of HPV-51.

The analysis of codon usage bias in HPV-51 shows a slight preference for A/T-ending codons. We additionally computed RSCU values for 59 synonymous codons, indicating that the favored codons in HPV-51 (RSCU > 1) mainly terminate with A or T. Such bias is commonly associated with the interaction of mutational pressure and natural selection, reflecting the virus’s evolutionary adaptation to optimize its replication and survival within the host (Sharp and Li 1987). The preference for A/T-ending codons may confer several advantages to HPV-51. First, these codons might be more abundant in the human genome, allowing the virus to camouflage its gene expression patterns and evade the host immune response (Karlin and Burge 1996). Second, in genomes with high AT content, a relationship frequently exists between codons ending in A or T and increased efficiencies in transcription and translation processes (Aktürk Dizman 2024). Additionally, the ENC values for HPV-51 suggest a relatively low overall codon usage preference. Identical findings of low codon usage bias were reported in other viruses like Zika virus (Tao and Yao 2020), molluscum contagiosum virus (Nair et al. 2021), and invertebrate iridescent virus (Aktürk Dizman 2023). The ENC value is an indicator of codon bias, with higher values signaling reduced bias and lower values suggesting a more pronounced bias towards particular codons. Previous studies have shown that viruses with higher ENC values tend to exhibit increased adaptability (Jenkins and Holmes 2003). Furthermore, a lower codon usage bias, as indicated by higher ENC values, may allow HPV-51 to conserve energy during viral biosynthesis and diminish interference with host protein synthesis, enhancing its overall fitness within the host environment (Wright 1990).

The frequency of dinucleotides significantly influences the preferences for codon usage in various organisms. Dinucleotide abundance reflects codon usage bias, mutational tendencies, and the impact of selective pressure and compositional constraints. Within viral genomes, dinucleotides’ proportional distribution significantly influences codon usage patterns (Upadhyay and Vivekanandan 2015). In our research, we examined 16 different dinucleotide frequencies. Notably, dinucleotides ApA, CpG, and TpC were less common, whereas CpA and TpG were more frequently observed than others. CpG depletion is a common viral strategy to evade host immune detection (Karlin and Burge 1995). Natural selection drives this reduction in CpG-containing codons, as host defenses recognize unmethylated CpG sequences, triggering immune responses (Khandia et al. 2019). To evade these defenses, viruses often reduce their CpG content (Vetsigian and Goldenfeld 2009). The increase in CpA and TpG may be compensatory, balancing the reduced CpG levels (Rima and McFerran 1997). Comparing dinucleotide frequencies with RSCU values of HPV-51 reveals that trinucleotide usage often mirrors dinucleotide preferences. The abundance or scarcity of specific dinucleotides can significantly shape the overall codon usage bias (Karlin and Burge 1995). In summary, dinucleotide frequencies are foundational elements shaping codon usage bias, reflecting the complex interplay between viral evolutionary pressures and host interactions in HPV-51.

Then, to comprehensively understand the factors shaping codon usage in HPV-51, we conducted PR2 analysis, ENC plots, and neutrality analysis. These analyses revealed a slight bias in codon usage, indicative of the intertwined implications of mutational pressure and natural selection. Interestingly, natural selection has emerged as the primary factor influencing codon preferences within HPV-51. This phenomenon likely signifies the virus’s adjustment to its host’s cellular environment and ability to evade the host’s immune responses. This preference might optimize viral protein synthesis and facilitate viral persistence. Our results align with previous research emphasizing the significance of natural selection in molding viral codon usage (Shackelton and Holmes 2008).

Additionally, we conducted an analysis of translational selection in HPV-51. In HPV-51, the most preferred codons utilize approximately half of the optimal codon-anticodon base pairings available in the human isoacceptor tRNA Pool. This preference for specific codons might indicate evolutionary pressures that optimize translation efficiency and accuracy (Kanaya et al. 2001). Utilizing the most compatible codons with the available tRNA molecules can enhance translation rates and reduce errors during protein synthesis (Ikemura 1985). The presence of approximately half of the optimal codon-anticodon pairs in the human isoacceptor tRNA pool facilitates efficient and accurate protein synthesis, thereby contributing to the organism’s overall fitness. This preference for specific optimal codons is believed to result from co-evolution between codon usage and tRNA availability, ensuring an optimal balance between translational speed and accuracy (Akashi 2001).

The positive correlation between GC3s and AROMO values suggests an intriguing relationship between compositional bias and amino acid aromaticity. This correlation implies that an increased compositional bias towards GC-rich codons is associated with higher aromaticity in proteins. One possible explanation for this correlation could be that GC-rich codons tend to encode aromatic amino acids more frequently, leading to proteins with higher aromaticity. Additionally, the selection pressures that favor GC-rich codons might also indirectly select proteins with increased aromaticity due to functional advantages conferred by aromatic amino acids (Duret 2002). The lower GC codon content in HPV-51, which is influenced more significantly by natural selection, supports this explanation. The interplay between codon bias, compositional, and amino acid properties is complex and multifaceted. Further research is needed to explore the mechanisms underlying these correlations and to elucidate the functional implications of increased aromaticity in proteins.

Finally, we evaluated the adaptation of HPV-51 to its human host using CAI and RCDI values. The CAI value for HPV-51 was determined to be 0.72 ± 0.00, indicating a moderate alignment with human codon preferences. This suggests that the virus has undergone adaptive evolution to utilize the host’s translational machinery efficiently. The RCDI value for HPV-51 was close to 2, further supporting its moderate adaptation to the human host. The concept of codon adaptation is crucial for understanding how viruses interact with their hosts at the molecular level. Viruses often optimize their codon usage to adapt to the translational machinery of the host cell, facilitating efficient production and duplication of viral proteins (Sharp et al. 2005). A high CAI value signifies a strong adaptation to host codon usage, whereas a low value may indicate a lack of adaptation or evolutionary divergence from the host. The RCDI value provides additional insights into the adaptation process by measuring the extent of codon deoptimization. A higher RCDI value suggests that the virus has minimized the use of non-preferred codons, which can lead to slower translation rates and increased competition with host mRNAs for ribosomal machinery (Plotkin and Kudla 2011). Therefore, an RCDI value close to 2 for HPV-51 indicates that the virus has struck a balance between efficiently utilizing the host’s translational machinery and minimizing excessive competition with host mRNAs Our findings suggest that HPV-51 has undergone adaptive evolution to align its codon usage with human preferences moderately. This adaptation likely contributes to the virus’s ability to efficiently replicate and propagate within the human host, highlighting the importance of codon adaptation in viral-host interactions.

Our previous research analyzed codon preference in HPV-33 and HPV-58 (Tan et al. 2024). Now, we compare the codon preference results of HPV-51 with those of HPV-33 and HPV-58. All three types – HPV-33, HPV-58, and HPV-51 – show a preference for codons ending in A or T, with GC3s values below 35%. The analysis of RSCU of HPV-31 revealed 30 favored codons, 26 concluding with A/T nucleotides, while among the 26 favored codons in HPV-33 and HPV-58, 25 concluded with A/U. The mean ENC values were 44.19 for HPV-33, 46.90 for HPV-58, and 48.25 for HPV-51, all above 35, indicating a relatively low level of codon preference. HPV-33, HPV-58, and HPV-51 exhibit specific patterns in dinucleotide frequency, with lower frequencies of ApA, CpG, and TpC, and higher frequencies of CpA and TpG. Analyses, including the ENC plot, PR2, and neutrality analysis, suggest that natural selection is the primary factor influencing codon usage in HPV-33, HPV-58, and HPV-51. The RCDI values for HPV-33, HPV-58, and HPV-51 are around 2, indicating a similar degree of codon usage preference to humans. These comparisons suggest that HPV-33, HPV-58, and HPV-51 share similar codon preferences, indicating common evolutionary mechanisms and selective pressures influencing codon usage.

While our study provides valuable insights into HPV-51’s codon usage bias, it is not without limitations. The analysis is based on genomic sequences from the NCBI GenBank database, which may not capture all HPV-51 types or regional variations (Van Doorslaer et al. 2013). Future studies could benefit from analyzing additional HPV-51 genomes, from diverse geographic regions and clinical samples, to further elucidate its codon usage patterns and evolutionary dynamics (Xi et al. 2006). Our thorough examination of codon utilization bias in HPV-51 reveals evolutionary patterns, host adaptation strategies, and translational efficiency within human cells. These findings enhance our understanding of HPV-51’s persistence, pathogenicity, and potential implications for HPV-related diseases.