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Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.

ABSTRACT: Papillomaviruses (PV) comprise a large family of nonenveloped DNA viruses that include the oncogenic PV types that are the causative agents of human cervical cancer. As is true of many animal DNA viruses, PV are taken into the cell by endocytosis and must escape from the endosomal compartment to the cytoplasm to initiate infection. Here we show that this step depends on the site-specific enzymatic cleavage of the PV minor virion protein L2 at a consensus furin recognition site. Cleavage by furin, a cell-encoded proprotein convertase, is known to be required for endosome escape by many bacterial toxins. However, to our knowledge, furin has not been previously implicated in the viral entry process. This step is potentially a target for PV inhibition.

SUBMITTER: Richards RM

PROVIDER: S-EPMC1360554 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.

Richards Rebecca M RM Lowy Douglas R DR Schiller John T JT Day Patricia M PM

Proceedings of the National Academy of Sciences of the United States of America 20060123 5

Papillomaviruses (PV) comprise a large family of nonenveloped DNA viruses that include the oncogenic PV types that are the causative agents of human cervical cancer. As is true of many animal DNA viruses, PV are taken into the cell by endocytosis and must escape from the endosomal compartment to the cytoplasm to initiate infection. Here we show that this step depends on the site-specific enzymatic cleavage of the PV minor virion protein L2 at a consensus furin recognition site. Cleavage by furin ...[more]

PMID: 16432208

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Project description:UnlabelledDespite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.ImportanceFurin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

Project description:Human papillomaviruses (HPVs) consist of two capsid proteins: major capsid protein L1 and minor capsid protein L2. The L2 protein has been shown to be involved in intracellular trafficking events that lead to the deposition of the viral DNA into the nucleus. In this study, we investigate the role of HPV16 L2 residues 43-DQILQ-47 during intracellular trafficking in human keratinocytes. We demonstrate that the highly conserved amino acids aspartic acid, isoleucine, and leucine are involved with the intracellular trafficking of the virus. Amino acid substitution of the isoleucine and leucine residues with alanine residues results in a significant decrease in infectivity of the pseudovirions without any changes to the binding or internalization of the virus. The pseudovirions containing these substitutions exhibit an altered trafficking pattern and do not deposit the viral pseudogenome into the nucleus. Instead, these mutated pseudovirions display a lack of interaction with syntaxin 18, an ER SNARE protein, are unable to progress past the endoplasmic reticulum (ER) and are redirected to the lysosomes. The results of this study help to elucidate the role and potential involvement of the 43-DQILQ-47 sequence during intracellular trafficking, specifically during trafficking beyond the ER. IMPORTANCE High-risk types of human papillomaviruses (HPVs), such as HPV16, are highly associated with cervical, anogenital, and oropharyngeal cancers. The minor capsid protein L2 is essential for the intracellular trafficking of the viral DNA to the nucleus. This study investigates the role of amino acid residues 43-DQILQ-47 of the HPV16 L2 protein in the intracellular trafficking of the virus. Understanding how the virus traffics through the cell is a key factor in the development of additional preventative antiviral therapies. This study illustrates, through modification of the 43-DQILQ-47 sequence in pseudovirions, the importance of the 43-DQILQ-47 sequence in the trafficking of the virus beyond the endoplasmic reticulum.

Dataset Information

Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.

Publications

Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.

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