Analysis Of Human Papillomavirus Type Research Articles

Whole-Genome Sequence Analysis of Human Papillomavirus Type 18 from Infected Thai Women

Objective: The aim of this study was to attain molecular knowledge of human papillomavirus type 18 (HPV18) by sequencing the whole genome of HPV18 isolated from Thai women at various clinical stages of disease progression. Method: Our group analyzed 9 samples of whole-genome HPV18 in infected women ranging from normal to cervical cancer by PCR, a sequencing method and bioinformatics programs. Results: Phylogenetic analysis based on the whole genome showed that HPV18 samples were more closely related to the European and Asian-American type than the African type. The vaccine strain’s L1 nucleotide (US patent 5820870) showed a close relationship to the African type. However, our data cannot indicate the correlation between cytological data and nucleotide or amino acid variation. Conclusion: Our group cannot draw any inference between the clinical stage of disease progression and amino acid alterations as there were only 1 or 2 samples available for each clinical trial. However, we hope that these new data on the HPV genome, which are representative of the entire genome of HPV in Southeast Asia, can serve as basis data for future research on the pathogenesis of cervical cancer. Additionally, the second-generation HPV18 vaccines should be tested on both HPV18-L1 and HPV18-L2 for increasing potential protection.

Analysis of Human Papillomavirus Type 18 Load and Integration Status from Low-Grade Cervical Lesion to Invasive Cervical Cancer

The clinical value of viral load and integration testing for human papillomavirus (HPV) remains unclear. Data on HPV type 18 (HPV18) is limited. We examined the HPV18 viral load and integration status of 78 women with normal cervix or neoplasia. While the crude viral load appeared to increase with lesion severity, the association was not significant after normalization with sample cellularity. Unlike reports for HPV16, the amino-terminal 1 region of HPV18 E2 was most frequently (71.0%) disrupted, representing the best marker for integration. A substantial proportion (57.1%) of invasive cancers harbored only the episomal genome, thus jeopardizing the clinical value of integration testing. A large proportion (41.7%) of normal/low-grade lesions showed viral integration, suggesting that integration of HPV18 starts early and is unlikely to be a sole determinant for progression. Interpretation of viral load should take into account the form of HPV infection as single infections had significantly higher viral loads than coinfections (P = 0.046). More data generated from routinely collected samples are warranted to verify the clinical value of viral load and integration testing. Viral load quantitation for HPV18 is premature for clinical use at this stage.

Analysis of Human Papillomavirus Type 16 (HPV16) DNA Load and Physical State for Identification of HPV16-Infected Women with High-Grade Lesions or Cervical Carcinoma

Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10(3) cells in normal samples up to 56,354 copies per 10(3) cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10(3) cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.

Methods for diagnosing papillomavirus infection.

The morphology of the lesion and the site in which the lesion is found are the initial clues in classifying papillomavirus-induced neoplasia. Human papillomavirus (HPV) types have limited site-specificity and differ in their association with benign or malignant neoplastic development. Cytopathology, electron microscopy, antigen detection and molecular hybridization all play a role in the armamentarium of diagnostic methods. Although nitrocellulose blotting procedures provide the most accurate and sensitive method for detecting and characterizing viral nucleic acid sequences, recent improvements in cytological hybridization methods allow for rapid detection of virus and analysis of HPV type directly in biopsied tissue and in cervical smears. In particular, these in situ hybridization procedures facilitate retrospective studies of stored specimens.

Quantitative analysis of human papillomavirus type 16 in cervical neoplasm: A study in Chinese population

Background: Human papillomavirus (HPV) infection was recognized as a major causal factor for the development and progression of squamous intraepithelial lesions (SIL). It is possible to use HPV test for the detection of cervical lesions as an adjunct to cervical cytology. Objectives: To evaluate the relation between HPV 16 viral load and the severity of cervical lesions in a Chinese population. Methods: Study population was recruited from the colposcopy and general outpatient clinic. The presence of HPV 16 E6 and E7 in cytological specimens was detected using HPV 16 specific polymerase chain reaction (PCR). The viral load in the specimens that were positive for HPV 16 specific PCR, was quantified by using real-time PCR assay. Results: The study recruited 394 women, in which 148 were high-grade SIL (HG-L), 121 were low-grade SIL (LG-L) and 125 were Normal. Sufficient DNA integrity was proven in 347 samples. Among 121 positive cases for HPV 16, 70 were HG-L, 34 were LG-L and 17 were Normal. Using quantitative real-time PCR, the percentages of samples with greater DNA copies were found to increase with the severity of diseases. There was also a significant difference in DNA copies among the three groups (HG-L versus Normal, p < 0.001; HG-L versus LG-L, p < 0.001). Area under receiver operating characteristic (ROC) curve of the HG-L versus LG-L and Normal was 0.836 indicating that quantitative PCR had a good diagnostic value in differentiating HG-L from the LG-L and Normal groups. Conclusions: Our data suggested HPV 16 viral load was significantly related to the severity of cervical lesions. Evaluation of viral burden could be a potential clinical tool in management of cervical lesions.

Analysis of Human Papillomavirus Type 16 E7-Specific T Cells in the Circulation of Patients with Virally Infected Squamous Cell Carcinoma of the Head and Neck

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Immune Monitoring

Molecular analysis of human papillomavirus type 52 isolates detected in the genital tract of human immunodeficiency virus-seropositive and -seronegative women.

Human papillomavirus (HPV) type 52 DNA was detected in cervicovaginal lavage samples from 91 (12.4%) of 732 human immunodeficiency virus (HIV)-seropositive women and 23 (7.1%) of 323 HIV-seronegative women (P=.0004). HIV infection was an independent predictor for HPV-52 infection when controlling for age and sexual activity (odds ratio, 2.21; 95% confidence interval, 1.30-3.75: P=.003). We describe the genomic polymorphism of 114 HPV-52 isolates. Long control region (LCR) mutations defined 27 HPV-52 variants. Nearly 32% of HPV-52 isolates carried deletions in the LCR. E6 and E7 mutations defined 17 and 9 variants, respectively. Five nonsynonymous E6 mutations were clustered from amino acids 92 to 94, near the putative p53 binding area. White women were more frequently infected by the prototype strain than were women of African descent (P=.0001). The genetic diversity of HPV-52 should facilitate the investigation of the role of genomic variations in cervical disease.

A comparative analysis of human papillomavirus types 16 and 18 and expression of p53 gene and Ki-67 in cervical, vaginal, and vulvar carcinomas

Objective This study aimed to investigate the correlation between HPV positivity, p53 overexpression, and cell proliferative activity in cervical, vaginal, and vulvar squamous cell carcinoma. Methods Sixteen vaginal and 31 vulvar squamous cell carcinomas were examined retrospectively for overexpression of p53 gene and Ki67 antigen by immunohistochemistry and for the presence of HPV types 16 and 18 DNA using a polymerase chain reaction (PCR) method. The results were compared with those obtained from 40 cervical squamous cell carcinomas. Results HPV type 16 or 18 DNA was detected in 21 (52.8%) of 40 cases of cervical carcinomas and p53 overexpression in one (2.5%), while HPV DNA sequences were detected in seven (43.7%) of 16 cases of vaginal carcinoma and p53 overexpression in three (18.7%). With regard to vulvar carcinoma, HPV was harbored in four (12.8%) of 31 cases and p53 overexpression in 19 (61.2%). These results indicated statistically significant inverse correlations between HPV positivity and p53 overexpression ( R = −0.999, P < 0.0001). Overexpression of Ki-67 was detected in 28 (70.0%) of 40, 12 (75.0%) of 16, and 21 (67.7%) of 31, cervical, vaginal, and vulvar carcinomas, respectively. There was no significant difference among the three groups. Conclusions In cervical carcinoma, HPV types 16 and 18 might play a common causal role, and in vulvar carcinoma, p53 gene mutations might be a main causal factor for carcinogenesis. Vaginal carcinoma, on the other hand, is considered to have transitional characteristics between cervical and vulvar carcinoma.

Mutational analysis of human papillomavirus type 16 major capsid protein L1: the cysteines affecting the intermolecular bonding and structure of L1-capsids

Human papillomavirus 16 major capsid protein L1 (composed of 505 amino acids (aa) including 12 cysteines) assembles by itself into virion-like icosahedral particles (L1-capsids), each of which is dissociated into 72 pentameric capsomeres when intermolecular disulfide bonds are disrupted. To identify the cysteines affecting the bonding and the structural integrity of the L1-capsids, we constructed a series of L1 mutants with substitution of serine for cysteine, which were expressed from recombinant baculoviruses in the insect Sf9 cells. From infected cells, the self-assembled L1-capsid fractions were purified by CsCl-equilibrium centrifugation and examined for velocity sedimentation profiles, for the presence of intermolecular bonding by SDS–PAGE with or without a reducing agent, for morphology under an electron microscope, and for susceptibility to trypsin digestion. Mutants C175S (C at aa 175 was replaced with S) and C185S were sedimented in sucrose-density gradients slightly slower than the wild type (WT) capsids, and mutant C428S stayed near the top as WT-capsomeres did. In the nonreducing SDS gel, where WT-capsids were separated into two bands of L1-trimers and L1-dimers, the C175S-trimer band was not detected, the C185S-dimer band was much less dense, and the C428S-trimer and C428S-dimer bands were not detected. Thus, it seems likely that C175, C185, and C428 are involved in L1 trimerization, in L1 dimerization, and in both, respectively. Morphologically, the C175S, C185S, and C428S fractions appeared to consist mostly of heterogeneous rod-shaped tubules, of smaller spherical particles, and of only capsomeres, respectively, whereas C102S, C229S, and C379S resembled WT. The C161S, C175S, C185S, C229S, C379S, and C428S capsids were more sensitive to degradation caused by trypsin than WT. The results indicate that C175, C185, and C428 are required for the normal assembly of L1-capsids through trimerization and dimerization of L1 bound by the intercapsomeric disulfide bonds between cysteines, and that C161, C229, and C379 are necessary for the integrity of L1-capsids probably through intramolecular bonding.

Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens.

Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process.

Analysis of human papillomavirus type 16 E6 variants in relation to p53 codon 72 polymorphism genotypes in cervical carcinogenesis.

This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.

Cervical cancer in older women: A molecular analysis of human papillomavirus types, HLA types, and p53 mutations: Gostout B.S.; Podratz K.C.; McGovern R.M.; Persing D.H. USA Am J Obstet Gynecol 1998 179/1 (56–61)

Cervical cancer in older women: A molecular analysis of human papillomavirus types, HLA types, and p53 mutations: Gostout B.S.; Podratz K.C.; McGovern R.M.; Persing D.H. USA Am J Obstet Gynecol 1998 179/1 (56–61)

Cervical cancer in older women: A molecular analysis of human papillomavirus types, HLA types, and p53 mutations

OBJECTIVE: The purpose of this study was to evaluate cervical cancers in older women to determine whether they differed from tumors in younger women with respect to human papillomavirus types, frequencies of p53 mutations, and presence of a proposed high-risk HLA-DR2 haplotype. STUDY DESIGN: Cervical tissue was obtained from women undergoing surgical treatment of in situ or invasive carcinoma of the cervix. Viral and genomic deoxyribonucleic acid was extracted. The presence of human papillomavirus deoxyribonucleic acid was detected by polymerase chain reaction amplification. Viral subtypes were assigned by means of a combination of type-specific amplification and automated sequencing of the L1 region. The presence of p53 mutations was evaluated by direct sequencing of exons 5 through 9. The HLA-DR locus was screened for the presence of the high-risk DRB1*1501 allele by means of selective polymerase chain reaction amplification followed by agarose gel electrophoresis of HLA-DR2 types. RESULTS: Tumors from 39 women 62 to 85 years old were analyzed. Tumors from 104 younger women formed a reference group. Human papillomavirus 16 was found in 41% and 54% and human papillomavirus 18 was found in 10% and 12% of the tissue samples from older and younger women, respectively. The overall distributions of human papillomavirus types did not differ statistically between the groups. One of the 25 older patients tested had a p53 mutation. This tumor also had a positive test result for human papillomavirus 18. The DR*1501 allele was present in 33% of the older patients and 28% of the younger patients. The expected frequency of this allele in white Americans is 19.8%. The increased frequency of this allele among both older and younger women with cervical cancer was statistically significant ( P < .05). CONCLUSIONS: We hypothesized that cervical cancer in older women might differ from that in younger women with respect to human papillomavirus types, natural host immunity, or the frequency of nonviral origins of the cancer. The findings show, however, that tumors from older women are extremely similar to those from younger women with respect to the human papillomavirus types present and the infrequent occurrence of p53 mutations. In addition we found that an HLA-DR allele that is associated with a risk of cervical cancer in younger women is also associated with risk in older women. These findings are most consistent with a model similar to that in younger women but with an unusually long latency for the transforming effect of the virus in some hosts. (Am J Obstet Gynecol 1998;179:56-61.)

Mutational analysis of human papillomavirus type 11 E5a oncoprotein

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.

Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells.

We have previously demonstrated that normal human mammary epithelial cells (MECs) are efficiently immortalized by human papillomavirus type 16 (HPV16) E6. HPV16 E6 binds to and induces p53 degradation in vitro and induces a marked reduction of p53 protein in MECs. Low-risk HPV6 E6 is defective for p53 binding and degradation in vitro but immortalized MECs at low efficiency. The HPV6 E6-immortalized MECs had markedly reduced levels of p53. To directly investigate whether the ability of HPV16 E6 to stimulate p53 degradation is required for E6-induced immortalization, a series of HPV16 E6 mutants were analyzed for the ability to bind and degrade p53 in vitro, induce a reduction in p53 levels in vivo, and immortalize MECs. We observed that one set of mutants efficiently immortalized MECs, caused a reduction in p53 levels in vivo, and degraded p53 in vitro. Other mutants immortalized MECs with low efficiency and either induced p53 degradation at low levels or were unable to induce p53 degradation in vitro; however, all of the immortal clones displayed low levels of p53. A third class of mutants did not immortalize MECs and failed to induce a reduction in p53 levels in vivo or degrade p53 in vitro. These results demonstrate that a reduction in p53 protein levels due to enhanced degradation is essential for MEC immortalization by HPV16 E6.

Mutational Analysis of Human Papillomavirus Type 16 E6 Protein: Transforming Function for Human Cells and Degradation of p53 in Vitro

The E6 oncoprotein of human papillomavirus type 16 (HPV 16) [151 amino acids (AA) long] contains four metal-binding motifs, C-X-X-C, and is postulated to form two 29-AA finger-like structures in the N-terminal and C-terminal halves, which mediate degradation of p53 and binding to p53, respectively. We constructed a series of E6 mutants with single-AA substitutions in these finger regions (AAs 34-62 and 107-135) and examined their transforming function for human embryonic kidney (HEK) cells in conjunction with HPV 16 E7 and their interaction with human p53 in vitro. The mutants with substitution of L for F-37, G for L-50, S for Y-54, and P for L-110, which did not transform HEK cells, showed markedly lowered activity to direct degradation of p53. The mutants with substitutions of G for R-39, G for V-42, G for Y-43, L for F-47, and G for V-53 lost the transforming function, but they could mediate degradation of p53 at levels comparable to the activities of the wild-type and transforming mutants. Like the wild type, all of the E6 mutants were localized by immunofluorescence to the nuclei of human TS21B cells or monkey COS-1 cells, except for the E6 mutant with substitution of G for Y-43 whose expression was undetectable. The levels of E6 mutants metabolically labeled in COS-1 cells were comparable to those of the transforming E6s. The data indicate that E6-directed degradation of p53 is necessary but not sufficient for HPV 16-mediated transformation of HEK cells.

Analysis of Human Papillomavirus Type 16 Variants Indicates Establishment of Persistent Infection

Sequence differences in the noncoding region of the human papillomavirus type 16 (HPV-16) genome were displayed using single-stranded conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified material. Two variants accounted for 50%-70% of all HPV-16 variants from 3 cohorts in Seattle. Seventy subjects who were repeatedly HPV-16 DNA-positive over 2-8 4-monthly visits showed an identical SSCP pattern at every visit. Only 10%-20% of the specimens showed evidence of infection by multiple variants when assessed by SSCP. However, cloning and sequencing of the PCR products revealed a substantially higher proportion of specimens with > 1 variant. Sequencing many clones from each specimen confirmed that 1 major variant seemed to predominate over time, whereas minor variants appeared more transient. These results suggest that HPV-16 establishes a persistent infection in which a single variant predominates: coinfection with addition HPV-16 variants results in a minor population of HPV-16 genomes.

Analysis of human papillomavirus type 16 E5 oncogene expression in vitro and from bicistronic messenger RNAs.

During a human papillomavirus infection (HPV), a set of spliced messenger RNAs (mRNA) are produced from the coding strand of the 8-kilobase virus DNA genome. These mRNAs have been isolated from cervical intraepithelial neoplasia biopsies, tumor biopsies and HPV type 16 (HPV-16)-containing cell lines, and subsequent analyses have shown that the different mRNAs are obtained by often complex splicing events. In the present study we have investigated the coding capacity of the HPV-16 E5 open reading frame (ORF) in an in vitro translation system and by the expression of a HPV-16 E5-CAT fusion gene from bicistronic messenger RNAs derived from polycistronic mRNAs which have previously been identified by others. Our results show that the HPV-16 E5 protein synthesis can only be initiated from the authentic ATG codon in the presence of E2 ORF translation. The E5 gene is found to be expressed only from a bicistronic mRNA with an E2 ORF upstream of the E5 gene indicating that the E5 protein is synthesized coordinated with the E2 protein. The supposition that the E5 protein is synthesized early in infection is discussed.

Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.

Functional analysis of human papillomavirus type 16 E7 by complementation with adenovirus E1A mutants.

Functional analysis of human papillomavirus type 16 E7 protein by complementation with adenovirus E1A mutants in baby rat kidney cells has shown that the retinoblastoma gene product (RB)-binding region of E7 can substitute in trans for that of E1A. An N-terminal E7 mutant was unable to complement an E1A mutant unable to bind p300, indicating that the two mutants were defective for functionally equivalent activities. E7 proteins with mutations within the RB-binding region were also unable to complement either the non-p300-binding E1A mutant or the N-terminal E7 mutant, suggesting that these mutations affect more than just RB binding.