Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

Abstract

Human papillomaviruses (HPVs) are a large group (Ͼ100 types) of small DNA viruses that replicate in keratinocytes of squamous epithelia. HPV infections produce hyperproliferative warts that are in most instances benign. A small subset of HPV types, however, form lesions on the skin, and on the oropharyngeal-and anogenital-tract mucosae, that have a significant risk of malignant transformation. The most common cancer attributable to infection with the high-risk HPV types is cancer of the uterine cervix (35). Despite the differences in pathogenesis between virus types, their life cycles are similar (10), beginning with infection of keratinocytes within the basal cell compartment of squamous epithelia. Here the HPV genome is replicated as a low-copy-number (50 to 100 copies per cell) episome in synchrony with the replication of the host cell genome, a process that requires HPV E1 and E2 functions. HPV early proteins E6 and E7 act to expand the population of HPV-infected keratinocytes once they migrate up from the basal layer, by stimulating cell cycle entry and cell survival. The virus then utilizes the host cell's replication machinery, which has been activated in these cells, to amplify the HPV DNA to many thousands of copies per cell during the vegetative stage of the life cycle. Finally, the capsid proteins L1 and L2 are produced, and new progeny are assembled in the most superficial cells prior to their release from the highly differentiated squames.A major protein produced during the HPV life cycle is the E4 protein. It is expressed as an E1^E4 fusion protein from spliced transcripts formed between the N terminus of the E1 open reading frame and almost the complete open reading frame of E4 (21). The precise function of E4 has not been defined, but loss of expression of the full-length E1^E4 polypeptide has a severe adverse effect on viral genome amplification of HPV type 16 (HPV16), HPV18, and HPV31 following introduction of mutant genomes unable to support E1^E4 expression into keratinocytes and subsequent induction of cellular differentiation (20,38,39). Failure to complete the vegetative stage of the virus life cycle is also the outcome of loss of E1^E4 expression in rabbit papillomas induced by a mutant cottontail rabbit papillomavirus genome (22). These studies suggest that E4 function is necessary for efficient vegetative replication of the virus, a hypothesis supported by coincidence between onset of viral genome amplification and induction of high-level E4 production in natural papillomavirus infections (23).Examination of E4 activity in epithelial cell cultures has revealed diverse biological actions that perhaps imply a multifunctional role for this viral protein in the virus life cycle. These include disruption of ND10 body organization, which might be required for viral DNA replication, either by organization of viral replication centers or by inactivation of a host

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1^E4 is necessary for association with CDK–cyclin complexes and G2/M cell cycle arrest of keratinocytes, but is not required for differentiation-dependent viral genome amplification or L1 capsid protein expression

“…Induction of cells already returned to S phase by E7 to arrest in G2/M could stimulate maintenance of a pseudo S phase-like state of the infected cell, and indeed, cell cultures that have been induced to arrest in G2/M by E4 show evidence of continuous rereplication of cellular DNA (Knight et al, 2004; Nakahara et al, 2002). However cellular rereplication is not always a consequence of E4 induced G2/M arrest and in some instances cellular DNA synthesis is al so inhibited, by E4 blocking the formation of pre-replication complexes at the cellular origin of replication (Knight et al, 2004; Roberts et al, 2008). An E4 mediated G2/M arrest of the cell cycle in replication-activated cells may therefore inhibit competing replication of the host genome so that the virus can have exclusive access to the host’s biosynthetic machinery for high-level synthesis of the viral DNA (Davy et al, 2002; Knight et al, 2004).…”

“…These include the suppression of cellular DNA synthesis through inhibition of chromosomal replication origin licensing (Knight et al, 2004; Roberts et al, 2008), the inhibition of the G2-to-Mtransition of the cell cycle (Davy et al, 2002; Knight et al, 2004; Nakahara et al, 2002), and the promotion of apoptosis by alteration of mitochondrial function (Raj et al, 2004). The viral protein can have a destabilizing effect upon the keratin cytoskeleton (Doorbar et al, 1991; Roberts et al, 1993; Roberts et al, 1997) and on the cornified envelope (Bryan and Brown, 2000), and also induces the redistribution of the promyelocytic leukaemia protein from sub-nuclear domains (Roberts et al, 2003).…”

Human Cytomegalovirus Protein pUL117 Targets the Mini-Chromosome Maintenance Complex and Suppresses Cellular DNA Synthesis

“…However, many viruses inhibit host DNA synthesis at critical stages of their replication cycle. Examples include adeno-associated virus (AAV) [41] , human papillomavirus (HPV) [42] , [43] , and herpesviruses [2] . How do these viruses block cellular DNA synthesis in an S-phase like environment?…”

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

“…By arresting cells in S phase, viral DNA synthesis is facilitated by the cellular DNA replication machinery; however, many DNA viruses also block cellular DNA synthesis for productive infection (40)(41)(42). Autonomous parvovirus MVM has been reported to inhibit host cell growth through p53-dependent inhibition of cyclin A, and the large nonstructural protein NS1 plays a key role in inhibiting host cell DNA synthesis (43)(44)(45).…”