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Similarity in genome organization between Molluscum contagiosum virus (MCV) and vaccinia virus (VV): Identification of MCV homologues of the VV genes for protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3β-hydroxysteroid dehydrogenase
Abstract
Molluscum contagiosum virus (MCV) and vaccinia virus (VV) are serologically unrelated poxviruses with a disparate genome composition (MCV, 66% G+C; VV, 33% G+C). Molecular studies of MCV have been hindered by the inability to propagate the virus in cells cultured in vitro. We sequenced 7765 bp of MCV DNA cloned from four widely spaced regions throughout the MCV genome and identified a total of 11 potential open reading frames (ORF), designated CX1-11. These include MCV homologues of the VV genes encoding protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3beta-hydroxysteroid dehydrogenase. The position and orientation of the MCV ORFs was collinear to the VV genome, with the exception of the region around ORF CX11 which is inverted in the MCV genome.
Supplementary resources (4)
Nucleotide Sequence
July 1995
Nucleotide Sequence
July 1995
Nucleotide Sequence
July 1995
Nucleotide Sequence
July 1995
... 1,8,10,11 Even though MCV is serologically distinct from VV, the sequencing of MCV has shown several homologues of the VV for signaling proteins, an RNA polymerase subunit, and structural proteins. 12 Hence, VV can be used as a surrogate to study MC proteins in vivo. 11 The genome of the MCV type I was decoded in 1997. ...
Background:
Molluscum contagiosum (MC) is an acute infection caused by the molluscum contagiosum virus (MCV) with a worldwide incidence of approximately 8,000 cases per 100,000 individuals annually. Greater than 90% of MC cases occur in the pediatric population, and affected adults are more likely to be younger or immunocompromised. MC has minimal inflammation initially; however, a strong inflammatory response can occur during resolution of the infection, termed the beginning of the end (BOTE). MC infections may last months to years, and it is hypothesized that persistent infections may be due to suppression of immunity by MCV proteins, thus affecting MC’s clinical progression.
Objective:
We reviewed the current proposed mechanisms of MCV immune evasion and discuss potential therapeutic options for MC treatment.
Methods:
A literature search was conducted using electronic databases (Pubmed, Google Scholar, Medline).
Results:
We compiled 18 original research articles and identified 11 proteins produced by MCV that are postulated to participate in evasion of host immunity through various molecular pathways. These proteins and/or their downstream pathways may be influenced by MC treatments in phase 3 development, including berdazimer gel 10.3% and VP-102 cantharidin, 0.7%.
Conclusion:
MCV is distinctive in evading immune surveillance by inhibiting or dampening several immune pathways via the production of viral proteins. The result is decreasing local inflammatory response which contributes to the prolonged survival of MCV in the epidermis. Persistent MC can be a nuisance for some patients and treatment may be desired. Currently, no treatment has been approved by the US Food and Drug Administration (FDA). Two approaches in the pipeline may affect the immune avoidance mechanisms; nevertheless, their exact mechanisms between the potential therapeutics and viral proteins remain enigmatic. J Drugs Dermatol. 2023;22(2):182-189. doi:10.36849/JDD.7230.
... Protein A44 shares 31% aa identity with human 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme needed for steroid biosynthesis [226,227,266]. Several other poxviruses such as molluscum contagiosum virus [267], fish lymphocystis virus [268] and avipoxviruses (fowlpox virus and canarypox virus) [269] also encode 3β-HSD. VACV A44 is an active 3β-HSD and is an early, non-essential protein that promotes virulence [163,223,224]. ...
The increasing frequency of monkeypox virus infections, new outbreaks of other zoonotic orthopoxviruses and concern about the re-emergence of smallpox have prompted research into developing antiviral drugs and better vaccines against these viruses. This article considers the genetic engineering of vaccinia virus (VACV) to enhance vaccine immunogenicity and safety. The virulence, immunogenicity and protective efficacy of VACV strains engineered to lack specific immunomodulatory or host range proteins are described. The ultimate goal is to develop safer and more immunogenic VACV vaccines that induce long-lasting immunological memory.
... Molluscum contagiosum 1 and 2 are related to orthopox and parapox viruses and share some homology with vaccinia. 1,2 Nitric oxide (NO) has been shown to have antiviral effects in DNA, RNA, enveloped and encapsidated viruses. 3,4 It reduces replication of herpes simplex type 1, 5 vaccinia and the orthopox ectromelia virus, 6 murine coronavirus 7 and in vitro, but not in vivo, has antiviral properties for avian reovirus infection. ...
A double-blind, group-sequential clinical trial of acidified nitrite was performed to demonstrate the efficacy of this nitric oxide donor in treating molluscum contagiosum. Subjects received either 5% sodium nitrite co-applied with 5% salicylic acid under occlusion, or identical cream with 5% salicylic acid, omitting sodium nitrite. Active and control treatment groups were well matched for the number and duration of lesions and made a similar number of applications. We found a 75% cure rate in the active treatment group and 21% cure with control treatment (P = 0.01). The mean time to cure was 1.83 months. Staining of the skin and irritation were frequent side-effects but did not prevent successful treatment.
... This is in contrast to VV, which causes a general infection throughout the body and provokes a much greater antibody response [Buller and Palumbo, 1991]. Furthermore, no cross-reactivity was found between the anti-VV and the anti-MCV sera, despite the existing homology at the amino acid level between antigenic VV proteins and their MCV counterparts [Blake et al., 1991; Sonntag et al., 1995; Douglass et al., 1996; Martin-Gallardo et al., 1996; Senkevich et al., 1996 Senkevich et al., , 1997 Moratilla et al., 1997]. On the other hand, the MCV genome encodes a number of speci®c proteins that presumably interact with the host immune system [Senkevich et al., 1996 [Senkevich et al., , 1997 Moratilla et al., 1997]. ...
Molluscum contagiosum virus (MCV) lesions from Spanish human immunodeficiency virus (HIV)-negative patients were clinically examined and analyzed for virus detection and typing. In a study of 147 patients, 97 (66%) were children under 10 years, of whom 49% had atopic dermatitis. MCV lesions were morphologically indistinguishable among the different age groups, but atopic patients presented larger lesions compared with patients without the disorder. In adults, lesions were observed mainly on the genitals. MCVI was the predominant subtype. The deduced MCVI/MCVII ratio (146:1) was much higher than that found in other geographical areas. Protein preparations of the virus-induced lesions were immunoblotted with sera from 25 MCVI patients. The host-serum antibody response was weak and variable, although no significant differences were found between atopic and nonatopic patients. Three immunoreactive proteins of 74/80, 60, and 35 kDa were detected in almost all the analyzed sera. The 35 and 74/80-kDa proteins were virus specific, whereas the 60-kDa protein band was composed of a mix of human keratins. Immunoblotting of MCV lesions and vaccinia virus-infected cell extracts with either MCV patient serum or a rabbit antiserum against vaccinia virus showed no cross-reactivity of these two human poxviruses at the antigenic level.
Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.
A library of molluscum contagiosum virus (MCV) transferred into the cowpox vector expression system was screened with 12 sera
from molluscum patients. Two recombinant proteins of 70 and 34 kDa were detected by immunoblotting and mapped to the open-reading
frames MC133L and MC084L, respectively. Consensus sites were found between the C-terminus of the 70-kDa MCV protein and the 14-kDa fusion protein
of vaccinia and variola virus, and between the 34-kDa MCV protein and the 37.5-kDa viral membrane-associated protein of vaccinia
and variola virus. Rabbit antisera against these two proteins were prepared. An immunofluorescence study demonstrated that
the 70- and 34-kDa proteins were predominantly expressed on the surface of recombinant virus- infected HeLa cells, indicating
the potential to be inserted into the membrane. On immunoelectron microscopy, antiserum against 70-kDa protein showed significant
labeling of the MCV membrane, while the antiserum against 34-kDa protein failed to do so.
Over the course of time poxviruses have acquired or "captured" numerous homologues of cellular genes and incorporated them into their large DNA genomes. With more poxvirus genome sequencing data becoming available, the number of newly discovered poxviral cellular homologues is constantly increasing. A common feature of these genes is that they are nonessential for virus replication in vitro and they confer selective advantages in dealing with host cell differentiation and immune defense mechanisms in vivo. Poxviral cellular homologues are reviewed in this synopsis considering the specific viral habitats of different poxviruses and the immune defence capabilities of their respective hosts. Possible mechanisms of cellular gene acquisition by poxviruses as suggested by the analysis of mobile genetic elements in large DNA viruses are discussed. The investigation of poxvirus homologues of cellular genes is essential for our understanding of the mechanisms that regulate virus/host interactions on the cellular level and the host response against infection.
Renewed interest in molluscum contagiosum virus has been stimulated by the availability of the entire genomic sequence and the identification of more than 160 putative genes, some of whose functions are now under analysis. Important findings have been generated by the recognition that certain viral proteins have anti-chemotactic and anti-apoptotic properties. Other advances include the characterization of two recombinant immunoreactive proteins, and the publication of the first seroepidemiological survey of the general population. Recent clinical studies indicate that some of the newer antiviral agents and physical treatment modalities offer significant benefits for the treatment of recalcitrant infections in the immunosuppressed.
Vaccinia virus open reading frame (ORF) SalF7L has 31% amino acid identity to human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD). Here we show that SalF7L encodes an active 3 beta-HSD, by the conversion of pregnenolone to the steroid hormone progesterone. The gene is transcribed early during infection into a 1.4 kb mRNA from an initiation site 12 bp upstream of the ORF. An antiserum raised against bacterially expressed SalF7L immunoprecipitated a 38 kDa polypeptide from infected cells, but not from mock infected cells or from cells infected with a mutant virus from which the SalF7L ORF had been removed. Deletion of the gene had no effect on virus replication in CV-1 cells in culture, yet the deletion mutant was attenuated when intranasally inoculated into mice. This steroid hormone synthesizing enzyme is a novel type of virus virulence factor.
The gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized. An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome. Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes. One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation. The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit. Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions. RNA analyses indicated that the 5'-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence.
We present the first nucleotide sequence data for molluscum contagiosum virus (MCV), an unclassified poxvirus. A 2,276-bp XhoI fragment from a near left-terminal fragment of MCV subtype I (MCVI) and a 1,920-bp XhoI fragment from the corresponding locus of MCV subtype II (MCVII) were sequenced and analyzed for open reading frames (ORFs). A large, complete ORF of 1,167 bp was present in both fragments. The putative polypeptide has a calculated molecular mass of 43 kDa (p43K protein) and was shown to have a high degree of homology to the vaccinia virus p37K major envelope antigen (40% amino acid identity and 22% conservative changes). The nucleotide content of the MCV fragments sequenced was 66% G or C. The codon usage within the gene for p43K reflected this high G + C content, with position 3 of codons being predominantly G or C (82 and 87% for MCVI and MCVII, respectively). The MCV p43K-encoding gene has motifs immediately upstream which are similar to those required for vaccinia virus late gene expression. The location and direction of transcription of the MCV p43K-encoding gene were equivalent to those of the vaccinia virus p37K gene, revealing similarity in genetic organization between MCV and vaccinia virus. Another, incomplete ORF was identified downstream of the p43K-encoding gene in both MCVI and MCVII. The sequence immediately upstream of this ORF overlapped the termination codon of the p43K-encoding gene and contained a motif which had homology to the derived consensus sequence for vaccinia virus early gene promoters.
The genomes (188 kbp) of the prototype Molluscum contagiosum virus type 1 (MCV-1) and a variant strain (MCV-1v) were characterized by construction of the physical maps of the viral DNA for the restriction enzymes BamHI, ClaI, EcoRI, and HindIII using a defined gene library harboring the DNA sequences of the MCV-1 genome and by DNA-DNA hybridizations. It was found that the genomes of both MCV strains are identical, with the exception of very few changes in the DNA fragmentation patterns of restriction endonuclease BamHI as a consequence of naturally occurring nucleotide exchanges in the genome of the variant strain. Detailed hybridization experiments revealed the existence of repetitive DNA sequences, which are located within the terminal regions of the viral genome at the map coordinates 0 to 0.027 and 0.973 to 1.
By sequencing a fragment of 7351 bp the fowlpox virus thymidine kinase gene has been found to map to a position within the equivalent of the vaccinia HindIII I fragment. The deduced gene arrangement in fowlpox virus is I3, X, TK, I5, I6, I7, I8, G1, indicating that the homologue of the vaccinia I4 gene has been replaced by two genes X and TK. The non-essential TK gene has therefore replaced another non-essential gene, I4 (the ribonucleotide reductase large subunit) in this region. The X/TK insertion in fowlpox virus is precisely flanked by direct repeats of 15 bp suggesting that the translocation event may have involved transposition. The % identities between the fowlpox virus and vaccinia virus proteins ranged between 58.5% and 31.3%.
DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease digestion, identifying three virus subtypes. The structural features of MCV DNA are typical of poxviral DNA. Physical maps of cleavage sites for BamHI, CIaI, and HindIII were constructed for single isolates of each subtype. These differ extensively, indicating the independence of the three subtypes. However, they are closely related, as determined by molecular hybridisation and nucleotide sequence analysis, and their genomes are essentially colinear. There is marked geographical variation in the relative incidence of MCV I and II, whilst MCV III is uniformly rare.
mRNA made in eukaryotic cells typically has a 3' poly(A) tail that is added posttranscriptionally. To investigate mechanisms by which 3' poly(A) is formed, we identified the genes for the two vaccina virus-encoded polypeptides, VP55 and VP39. Primer-dependent polyadenylation activity was associated exclusively with purified VP55-VP39 heterodimer, which, although stable to column chromatography and glycerol gradient sedimentation, was readily dissociated by antibody to an N-terminal peptide of VP55. Poly(A) polymerase activity was associated with immunopurified VP55, but not with immunopurified or chromatographically purified VP39. VP39 was, however, required for the formation of long poly(A) molecules, in conjunction with either purified VP55 or low concentrations of the heterodimer, and was shown to bind free poly(A). Thus, a catalytic polypeptide and a dissociable poly(A)-binding stimulatory factor each contribute to poly(A) tail formation. No prokaryotic or eukaryotic homologs of either polypeptide were detected in sequence data bases, consistent with the absence of previously reported poly(A) polymerase genes from any source.
Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression. Synthetic oligonucleotides, based primarily on the sequence of the 7·5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus. The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for β-galactosidase expression. The relative amounts and precise 5′ ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. Many promoters were tested for their ability to direct specific transcription in vitro. A generally good correlation was noted between measurements of promoter strength estimated by β-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro.
DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.
DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease cleavage, revealing two virus subtypes. Physical maps of cleavage sites for BamHI, ClaI and HindIII were constructed, and found to differ extensively between the two subtypes. MCV DNA was similar to Orthopoxvirus DNA with respect to size, terminal cross-linking and the presence of inverted terminal repetitions, but did not hybridize with vaccinia virus DNA. The genomes of the two MCV subtypes cross-hybridized and were colinear except for two small regions. There was sequence homology between DNA from corresponding map regions of the MCV subtypes but, in contrast to Orthopoxvirus DNA, no conservation of restriction sites. A synthetic oligonucleotide probe representing a conserved domain of epidermal growth factor, alpha-transforming growth factor and the vaccinia growth factor identified equivalent regions of both MCV genomes as having the potential to encode this domain. This locus is similar to the position of the vaccinia growth factor gene in vaccinia virus DNA. Thus MCV may induce epidermal cell proliferation and tumourigenesis by expression of an epidermal growth factor-like polypeptide.