Typing of human papillomaviruses in condylomata acuminata from Greece.

DNA samples from recurrent condylomata acuminata biopsies of Greek males and females were examined for the presence of human papillomavirus (HPV) DNA using high-stringency Southern blot hybridization analysis. Of the twenty-six biopsies, 25 were positive for the HPV 6/11-related DNA sequences, and when further analyzed with the polymerase chain reaction (PCR) the HPV-negative biopsy was also positive for HPV 6/11 DNA. Nineteen specimens were further characterized based on their Pstl restriction endonuclease hybridization pattern. Twelve biopsies were positive for HPV 6a, one biopsy was positive for HPV 11a, and one biopsy was positive for HPV 6c DNA. Three specimens contained HPV 6/11 related DNA that gave an unusual Pstl pattern, and one specimen appeared to represent a multiple HPV infection containing HPV 6/11- and HPV 31/35/39-related sequences. Finally, one sample contained a mixture of HPV 6a DNA and an HPV 6a-like genome. Biopsies were also taken from adjacent apparently normal tissue, 0.5 cm away from the lesion, in 19 of the patients. Only one of these was found to be positive for HPV 6a DNA by Southern blot analysis.

Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups.

Papillomaviruses are an ideal model system for the study of DNA virus evolution. On several levels, phylogenetic trees of papillomaviruses reflect the relationship of their hosts. Papillomaviruses isolated from remotely related vertebrates form major branches. One branch of human papillomaviruses (HPVs) includes an ape and two monkey papillomaviruses, possibly because the diversification of the viruses predated the separation of the infected-primate taxa. This hypothesis predicts that the root of the evolution of some if not all HPV types should point to Africa, since humans evolved from nonhuman primates in this continent. We tested this hypothesis and compared the genomic sequences of HPV type 18 (HPV-18) isolates from four continents. Diversity within HPV-18 correlates with patterns of the evolution and spread of Homo sapiens: HPV-18 variants, just like HPV-16 variants, are specific for the major human races, with maximal diversity in Africa. Outgroup rooting of the HPV-18 tree against HPV-45, which is closely related to HPV-18, identifies African HPV-18 variants at the root of the tree. The identification of an African HPV-45 isolate further reduces the evolutionary distance between HPV-18 and HPV-45. HPV-18 variants from Amazonian Indians are the closest relatives to those from Japanese and Chinese patients and suggest that a single point mutation in the phylogenetically evaluated genomic segment represents at least 12,000 years of evolution. We estimate that diversity within HPV-18 and probably within other HPV types evolved over a period of more than 200,000 years and that diversity between HPV types evolved over several million years.

The genetic drift of human papillomavirus type 16 is a means of reconstructing prehistoric viral spread and the movement of ancient human populations.

We have investigated the diversity of a hypervariable segment of the human papillomavirus type 16 (HPV-16) genome among 301 virus isolates that were collected from 25 different ethnic groups and geographic locations. Altogether, we distinguished 48 different variants that had diversified from one another along five phylogenetic branches. Variants from two of these branches were nearly completely confined to Africa. Variants from a third branch were the only variants identified in Europeans but occurred at lower frequency in all other ethnic groups. A fourth branch was specific for Japanese and Chinese isolates. A small fraction of all isolates from Asia and from indigenous as well as immigrant populations in the Americas formed a fifth branch. Important patterns of HPV-16 phylogeny suggested coevolution of the virus with people of the three major human races, namely, Africans, Caucasians, and East Asians. But several minor patterns are indicative of smaller bottlenecks of viral evolution and spread, which may correlate with the migration of ethnic groups in prehistoric times. The colonization of the Americas by Europeans and Africans is reflected in the composition of their HPV-16 variants. We discuss arguments that today's HPV-16 genomes represent a degree of diversity that evolved over a large time span, probably exceeding 200,000 years, from a precursor genome that may have originated in Africa. The identification of molecular variants is a powerful epidemiological and phylogenetic tool for revealing the ancient spread of papillomaviruses, whose trace through the world has not yet been completely lost.

Identification of a new transcriptional unit that yields a gene product within the unique sequences of the short component of the herpes simplex virus 1 genome.

The herpes simplex virus genome 1 consists of two unique stretches, long (UL) and short (U(S)), each flanked by inverted repeat sequences. The U(S) sequence has been previously reported to contain 12 open reading frames designated U(S)1 through U(S)12. This report demonstrates the existence of a 13th open reading frame within the U(S) sequence, designated U(S)8.5. The U(S)8.5 sequence is located between, and overlaps in part with, the domains of the U(S)8 and U(S)9 genes. Its transcription is initiated within the coding sequence of U(S)8, and its transcript decays earlier than that of U(S)8. On the basis of the size of its RNA (1.2 kb) and map position, it is likely that the U(S)8.5 transcript is 3' coterminal with the U(S)8 and U(S)9 mRNAs at the single polyadenylation signal which serves these genes. The nucleotide sequence of the U(S)8.5 open reading frame predicts that its product is a 151-amino-acid basic, hydrophilic polypeptide. To determine whether the U(S)8.5 encodes a protein, a sequence encoding 23 amino acids that contains an epitope reacting with a known monoclonal antibody to human cytomegalovirus protein was inserted in frame after the predicted fifth codon of the U(S)8.5 gene. The recombinant virus carrying this epitope induced the synthesis of a protein reactive with the monoclonal antibody in immunoblots. The tagged protein localized in nucleoli of cells infected with the recombinant virus.

Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.

Delineation of regulatory domains of early (beta) and late (gamma 2) genes by construction of chimeric genes expressed in herpes simplex virus 1 genomes.

The expression of the gamma 2 class of viral genes in cells infected with herpes simplex virus 1 requires viral DNA synthesis and functional viral products made earlier in infection. To identify the sequences required for gamma 2 gene expression, we constructed recombinant viruses in which regions of the thymidine kinase gene (tk), a beta gene normally expressed early in infection, were replaced by specific domains of a gamma 2 gene. The phenotypic attributes examined were (i) sensitivity or resistance of expression in cells exposed to sufficient phosphonoacetate to block viral DNA synthesis, properties of gamma 2 and beta genes, respectively; (ii) expression early in infection, a property of beta genes; and (iii) expression late in infection, a property of gamma 2 genes. We report that replacement of nucleotides -200 to +51 of tk with nucleotides -77 to +104 of the gamma 2 gene conferred upon tk all of the tested attributes of gamma 2 genes. The tk sequence in the 5' transcribed noncoding domain downstream of nucleotide +51 played no apparent role in the expression of the chimeric genes. Similarly, tk sequence downstream of -16 and gamma 2 sequence upstream of -12, when juxtaposed in correct orientations, yielded a chimeric gene that was poorly expressed. In contrast, the chimeric gene consisting of tk sequence upstream of -16 fused to gamma 2 sequence downstream of -12 had the attributes of both beta and gamma 2 genes in that it was expressed both early and late in infection and was partially resistant to phosphonoacetate. The capacity for expression late in infection encoded in the gamma 2 5' transcribed noncoding domain was observed in cells infected with a recombinant virus in which gamma 2 nucleotides +17 to +104 were inserted into the 5' transcribed noncoding domain of the tk gene. We conclude that whereas in the beta genes exemplified by the tk gene the regulatory domains are mainly upstream from nucleotide +51, the sequence(s) that confers gamma 2 regulation is downstream from the TATAA box.

The trans-activation of herpes simplex virus gene expression: comparison of two factors and their cis sites.

In herpes simplex virus-infected cells, gene expression is tightly regulated. In this review, we compare the properties of two trans-activating factors which regulate the expression of viral genes. The first, alpha trans-inducing factor (alpha TIF) is a structural component which induces the 5 alpha genes, the first set of genes transcribed after infection. Alpha TIF requires for induction a cis-acting site present in promoter-regulatory domains of all alpha genes. The cis site binds 2 host proteins, alpha H1 and alpha H2-alpha H3. These host proteins have a maximum bound molecular weight of 110,000 and 64,000, respectively. DNase 1 protection assays indicate that alpha H1 protects the entire cis site, whereas alpha H2-alpha H3 binds the 3' domain of the cis site. The methylation interference assays indicate that the contact points of alpha H1 and alpha H2-H3 are at the 5' and 3' termini of the cis site, respectively. Both proteins can bind to the cis site concurrently. Alpha TIF does not bind directly to DNA but was shown to be present in DNA-protein complexes. The binding of alpha TIF to these DNA-protein complexes requires the participation of alpha H1. In contrast to alpha TIF, the product of the alpha 4 gene, a protein 163,000 in apparent molecular weight binds to DNA directly and regulates genes both positively and negatively. The data indicate that alpha 4 protein can bind to at least 2 binding sites differing in nucleotide sequence and which can be present in promoters, across the transcription initiation sites, and in 5' transcribed non-coding sequences. The regulatory functions of the alpha 4 protein may reflect both the nature and location of the binding site. The biological implications of the viral trans-acting proteins are discussed.

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses.

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.

Virulence of and establishment of latency by genetically engineered deletion mutants of herpes simplex virus 1.

We report the results of studies on the biologic properties of seven deletion mutants of herpes simplex virus 1 (HSV-1). The genes deleted from six of these mutants map in the S component of HSV-1 DNA and include those specifying the alpha protein 47, the glycoproteins G and E, the viral protein kinase, and two proteins whose functions are not yet known (open reading frames US2 and US11). The seventh virus [HSV-1(F) delta 305] contained a 700-bp deletion in the thymidine kinase gene. The results of intracerebral inoculation of Balb/c mice indicated that all but one of the deletion mutants in the S component were significantly attenuated. The PFU/LD50 ratios for these mutants ranged from 10(4)- to 10(5)-fold higher than that of the wild-type, HSV-1(F). The PFU/LD50 for mutant R7032, from which the glycoprotein E gene had been deleted, was less than 100-fold higher than that of the parent virus. All of the mutants, with one exception, were able to establish latency in mice; the exception, HSV-1(F) delta 305, was able to establish latency in rabbits.

Activation of herpes simplex virus 1 gamma 2 genes by viral DNA replication.

The expression of gamma 2 or true gamma genes resident in the herpes simplex virus 1 genome requires functional products of genes expressed earlier in infection and viral DNA synthesis. To determine whether the requirement for viral DNA synthesis for the expression of gamma 2 genes reflects a trans-acting function of a product of one or a few genes made or activated during viral DNA synthesis or whether it reflects a cis effect of DNA synthesis at specific sites, 143 thymidine kinase (TK) minus cells were sequentially infected with a virus carrying a 700-bp deletion of the TK gene and 6 hr later with wild-type or recombinant viruses carrying an alpha-TK or a gamma 2-TK gene in either the presence or absence of viral DNA synthesis. These experiments indicated that the gamma 2-TK gene contained in an unreplicated viral genome was not expressed by trans-acting factors specified before and during DNA synthesis by the first infecting virus, and, therefore, the induction of gamma 2 genes appears to be mediated by a cis-acting function associated with viral DNA synthesis.

Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha 47 gene.

The five alpha genes, alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, are the first set of herpes simplex virus 1 genes to be transcribed and expressed in productively infected cells. We report here the construction of a viral recombinant from which all of the coding sequences of the alpha 47 gene were deleted. In addition to the alpha 47 protein, infected cell lysates did not contain detectable amounts of two polypeptide bands with apparent molecular weights of 18,000 and 21,000 which could be specified by a gene whose regulatory domain and 5' transcribed noncoding sequences overlap with the coding sequences of the alpha 47 gene. The alpha 47- virus grew as well as the wild-type parent virus in Vero, baby hamster kidney, and Rat-1 cell lines.

Regulation of herpes simplex virus 1 genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late (gamma 2) promoters.

This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518-528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests.