Do you want to know how Human Papillomaviruses hijack host machinery?

Benzyme Ventures
4 min readMay 5, 2021

Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking

Pengwei Zhang, Gabriel Monteiro da Silva, Catherine Deatherage, Christopher Burd, Daniel DiMaio (2018).

Summarised by Changin Oh

Original article —

Cellular membranes are the first line of host defense that physically block invading viruses. In other words, viruses must penetrate these membranes, to cause an infection. For example, Coronaviruses use their spike protein to pass through the host’s cellular membrane by virus-cellular membrane fusion. Alternatively , non-enveloped viruses use a lytic peptide to physically disrupt the membrane or form a large pore. All these different mechanisms allow viruses to translocate the viral genome into the cytoplasm or nucleus to initiate replication.

Human papillomaviruses (HPVs) are small non-enveloped viruses causing a majority of cervical cancers. The HPV protein shell comprises 360 molecules of L1 major capsid protein and up to 72 copies of the L2 minor capsid protein. The L2 protein plays an essential role in translocating the virus and viral DNA by interacting with the retromer complex. The retromer complex is a key component of the cellular sorting machinery. It recognizes specific membrane proteins and transports them to the appropriate destination. HPV hijacks this retromer complex to escape from the endosome into the Trans-Golgi network (TGN). Interestingly, retromer directly binds to conserved sites in the HPV L2 (Fig 1A). However, there is a topological challenge here: the incoming viruses are inside the endosome but the retromer remains outside (in the cytoplasm). Thus, this study started with an interesting question — what makes one end of the L2 protein of HPV protrude through the endosomal membrane into the cytoplasm to initiate this interaction with the retromer?

To answer this question, firstly, they compared DNA sequences of papillomavirus L2 proteins. All of them contain a stretch of basic amino acids (arginine and lysine), which resembles a class of peptides known as cationic cell-penetrating peptides (CPPs) near the retromer binding site (Fig 1A). For further experiments, the author used HPV pseudovirus (PsV) comprising capsid proteins (wild type L1, and wild-type or mutant L2) and a reporter plasmid (HcRed). HPV PsV is a standard tool for studying HPV cellular entry and neutralization due to its safety (PsVs are only capable of replicating once and lack the virulent components of the original virus) and efficiency (PsV can be easily and rapidly produced).

Secondly, to identify the function of the stretch of basic amino acids (L2 CPP), they replaced the stretch with six alanines or three arginines (3R) to disrupt the function of L2 CPP, and then infected the cells. These mutant PsVs lost their infectivity (Fig 1C). When they replaced the stretch with the cell-penetrating peptide domain of HIV Tat protein, the PsV recovered infectivity (Fig 1C). The cell-penetrating activity of the stretch was confirmed using a short fragment of protein combining with HPV16 L2 C-terminal peptide and green fluorescence protein (Fig 2).

Next, the role of the L2 CPP in HPV infection was dissected using confocal laser scanning microscopy. PsV with either wild type (WT) or 3R L2 can bind to cell surface receptors and enter the endosome in the host cell (Fig 3). However, wild type HPV PsV successfully escaped from the endosome by 8 hours post-infection (hpi) and arrived at the TGN by 16hpi, whereas the 3R mutant HPV PsV remained in the endosome even at 16hpi and could not arrive at the TGN (Fig 4). This is because the 3R mutant could not access the retromer during HPV infection (Fig 5). This shows that L2 CPP facilitates the cytoplasmic exposure of one end of L2 (Fig 6 and 7).

Endosomal escape of the virus, (by passing through the endosomal membrane), is a critical process in the life cycle of the virus. If the virus fails to escape from the endosome, it will be transferred to the endo-lysosome for degradation. In this study, they clearly demonstrated how HPV escapes from the endosome. The CPP in the HPV L2 protein enables the viral protein to pass across the endosomal membrane into the cytoplasm to bind the retromer, which then enables HPV to use the retrograde pathway. Also, they defined the intracellular biological role of CPP in its natural context. Overall, this study thoroughly examined and revealed the secret of CPP in a logical flow with solid data and good experimental controls. Their related publication is also very interesting if you would like to read it — they applied short amino acid fragments containing the CPP to inhibit HPV entry and replication, which is a new approach for antiviral drug development (links attached below).

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