The results presented in this paper confirm that HSV-1 virion DNA has an unusual structure, containing multiple nicks and gaps. Untreated virion DNA was as infectious as DNA whose nicks and gaps were filled in and ligated, suggesting that nicks and gaps are not required for virion DNA to be infectious.
We have been particularly interested in whether cells respond to unusual viral DNA conformations by signaling a damage response that could be antiviral. Using a novel assay based on the biochemical properties of the Klenow polymerase, we were also able to estimate that virion DNA contains, on average, 15 gaps per genome, consistent with published observations 9 , In this study, we estimated that the average size of gaps in virion DNA was approximately 33 nucleotides, long enough bind a single RPA complex but too short to activate ATR signaling.
Thus, the incoming genome does not appear to be a suitable substrate for ATR activation. Only a few other DNA viruses are known to package nicked and gapped genomes: pseudorabies virus PRV and Marek's disease virus MDV have nicks and gaps that are randomly distributed 64 , — 66 , while other DNA viruses contain nicks and gaps located at specific sites. For example, there are five major nicks in T5, which are thought to occur as a result of a virally encoded nicking enzyme and may play a role in the two-step transfer mechanism for ejecting DNA into its host 67 , — Like T5, T7 DNA also has single-strand interruptions at specific sites, but these are thought to be the product of premature terminase activity during packaging Little is known, however, about whether the structure of viral genomes plays a role in infectivity or stimulation of host DDR.
Cells have several different DDR pathways that could be activated during infection, and it is possible that HSV has evolved to utilize those pathways that are conducive to productive infection while preventing pathways that inhibit lytic infection. Taken together, these results suggest that the activation of DNA-PK is associated with the drop in infectivity, and we are intrigued by the mechanism by which this occurs.
This model, however, does not explain the observation that the absence of DNA-PK itself can rescue infectivity.
It is possible that DNA-PK, acting as part of the NHEJ pathway, promotes circularization of the viral genome, which has been correlated with establishment of latent or quiescent infection 77 , — The report by Jackson and DeLuca that the presence of ICP0 can inhibit circularization 77 may be consistent with this suggestion; however, further experimentation will be required to elucidate the relationship between NHEJ and circularization of viral genomes.
UL12 interacts with ICP8 to form a two-component recombinase capable of strand exchange 80 , We are intrigued by the possibility that UL12 and ICP8 work together to promote recombination-dependent replication by SSA and that this pathway leads to the production of concatemeric DNA that can be packaged into infectious virus In addition, we are currently exploring the possible involvement of cellular proteins in the stimulation of SSA in HSV-infected cells.
Our findings suggest that the presence of nicks and gaps in incoming DNA may result in the recruitment of a combination of cellular and viral proteins that stimulates a repair pathway that is beneficial to lytic replication, such as SSA. This process underscores the complex evolutionary relationships between HSV and its host. Published ahead of print 25 June National Center for Biotechnology Information , U.
Journal List J Virol v. J Virol. Sandri-Goldin, Editor. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address correspondence to Sandra K.
Weller, ude. Schumacher, 3M, St. Paul, Minnesota, USA. Received Jun 16; Accepted Jun All Rights Reserved. This article has been cited by other articles in PMC. Viruses and plasmids. Preparation of viral DNA. In vitro modification of virion DNA.
Measurement of nucleotide incorporation into DNA. Calculation of gap number and length. Infectivity assays. Pulsed-field gel electrophoresis.
Western blot analysis. Gene expression assay. Open in a separate window. FIG 1. Klenow polymerase strand displacement activity can be used to measure gap number and length. FIG 2. FIG 3. Filling in nicks and gaps did not affect infectivity. FIG 4. Treatment with mung bean nuclease destroys infectivity, confirming the presence of gaps.
Treatment with calf intestine alkaline phosphatase is tolerated. Treatment with Klenow polymerase abolishes infectivity. FIG 5. FIG 6. FIG 7. FIG 8. FIG 9. Footnotes Published ahead of print 25 June Infectious DNA from herpes simplex virus: infectivity of double-stranded and single-stranded molecules. Jacob RJ, Roizman B. Anatomy of herpes simplex virus DNA. Properties of the replicating DNA.
Size, composition, and structure of the deoxyribonucleic acid of herpes simplex virus subtypes 1 and 2. Frenkel N, Roizman B. Separation of the herpesvirus deoxyribonucleic acid duplex into unique fragments and intact strand on sedimentation in alkaline gradients. Wilkie NM. Replicating DNA of herpes simplex virus type 1. Intervirology 7 — [ PubMed ] [ Google Scholar ]. Ribonucleotides in newly synthesized DNA of herpes simplex virus.
Virology 61 — Studies on herpes simplex virus DNA: denaturation properties. Virology 55 — Biochemistry 21 — Replication of herpesvirus DNA.
IV: analysis of concatemers. Virology 94 —70 [ PubMed ] [ Google Scholar ]. In vitro repair of the preexisting nicks and gaps in herpes simplex virus DNA. Virology 76 — Lukashchuk V, Everett RD. EMBO J. Attenuation of DNA-dependent protein kinase activity and its catalytic subunit by the herpes simplex virus type 1 transactivator ICP0. Herpes simplex virus type 1 immediate-early protein vmw induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase.
PLoS Pathog. Abraham RT. DNA Repair Amst. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Cell 40 — Cimprich KA, Cortez D. Many proteins are known to be virion structural components, or to have regulatory roles, or to function in synthesis of virus DNA. Many, however, still lack an assigned function. Two classes of genetic entities necessary for virus DNA replication have been characterized: cis-acting sequences, which include origins of replication and packaging signals, and genes encoding proteins involved in replication.
Aside from enzymes of nucleotide metabolism, the latter include DNA polymerase, DNA binding proteins, and five species detected by genetic assays, but of presently unknown functions. Complete genome sequences are now known for the related alphaherpesvirus varicella-zoster virus and for the very distinct gammaherpesvirus Epstein-Barr virus.
Tightly adherent to the capsid is the tegument, which appears to consist of amorphous material. Loosely surrounding the capsid and tegument is a lipid bilayer envelope derived from host cell membranes. The envelope consists of polyamines, lipids, and glycoproteins. These glycoproteins confer distinctive properties to each virus and provide unique antigens to which the host is capable of responding.
A fascinating feature of herpesvirus DNA is its genomic sequence arrangement. Herpesviruses can be divided into six groups arbitrarily classified A to F. For those herpesviruses which infect humans group C, group D, and group E unique structures are demonstrable.
In the group C genomes, as exemplified by Epstein-Barr virus and the newly identified Kaposi's sarcoma herpesvirus, the number of terminal reiterations divides the genome into several well-delineated domains. The group D genomes, such as varicella-zoster virus, have sequences from one terminus repeated in an inverted orientation internally. Thus, the DNA extracted from these virions consist of two equal molar populations. For group E viral genomes, such as herpes simplex virus and cytomegalovirus, the genomes are divided into internal unique sequences whereby both termini are repeated in an inverted orientation.
Thus, the genomes can form four equimolar populations which differ in relative orientation of the two unique segments. The grouping of herpesviruses into sub-families serves the purpose of identifying evolutionary relatedness as well as summarizing unique properties of each member. The members of the alpha herpesvirus sub-family are characterized by an extremely short reproductive cycle hours , prompt destruction of the host cell, and the ability to replicate in a wide variety of host tissues.
They characteristically establish latent infection in sensory nerve ganglia. This sub-family consists of herpes simplex virus 1 and 2 and varicella-zoster virus. In contrast to the alpha herpesviruses, beta herpesviruses have a restricted host range. Their reproductive life cycle is long days , with infection progressing slowly in cell culture systems.
A characteristic of these viruses is their ability to form enlarged cells, as exemplified by human cytomegalovirus infection. These viruses can establish latent infection in secretory glands, cells of the reticuloendothelial system, and the kidneys. Finally, the gamma herpesviruses have the most limited host range. They replicate in lymphoblastoid cells in vitro and can cause lytic infections in certain targeted cells.
Latent virus has been demonstrated in lymphoid tissue. Epstein-Barr virus is a member of this sub-family. In addition, human herpesvirus 6 and 7 are probably best classified as a gamma herpesvirus. However, the latter has host range properties of the beta sub-family. Further studies will need to clarify the most appropriate classification of this virus. Kaposi's sarcoma herpesvirus is most closely related genetically to Epstein-Barr virus.
Replication of all herpesviruses is a multi-step process. Following the onset of infection, DNA is uncoated and transported to the nucleus of the host cell.
This is followed by transcription of immediate-early genes, which encode for the regulatory proteins. Expression of immediate-early gene products is followed by the expression of proteins encoded by early and then late genes. Assembly of the viral core and capsid takes place within the nucleus. This is followed by envelopment at the nuclear membrane and transport out of the nucleus through the endoplasmic reticulum and the Golgi apparatus.
Glycosylation of the viral membrane occurs in the Golgi apparatus. Mature virions are transported to the outer membrane of the host cell inside vesicles. Release of progeny virus is accompanied by cell death. Replication for all herpesviruses is considered inefficient, with a high ratio of non-infectious to infectious viral particles. A unique characteristic of the herpesviruses is their ability to establish latent infection.
Each virus within the family has the potential to establish latency in specific host cells, and the latent viral genome may be either extra-chromosomal or integrated into host cell DNA. Herpes simplex virus 1 and 2 and varicella-zoster virus all establish latency in the dorsal root ganglia.
Epstein-Barr virus can maintain latency within B lymphocytes and salivary glands. Cytomegalovirus, human herpesvirus 6 and 7, Kaposi's sarcoma herpesvirus and B virus have unknown sites of latency. Latent virus may be reactivated and enter a replicative cycle at any point in time. The reactivation of latent virus is a well-recognized biologic phenomenon, but not one that is understood from a biochemical or genetic standpoint.
It should be noted here that an anti-sense message to one of the immediate-early genes alpha-O may be involved in the maintenance of latent virus. Stimuli that have been observed to be associated with the reactivation of latent herpes simplex virus have included stress, menstruation, and exposure to ultraviolet light. Precisely how these factors interact at the level of the ganglia remains to be defined. It should be noted that reactivation of herpesviruses may be clinically asymptomatic, or it may produce life-threatening disease.
With the exception of cytomegalovirus retinitis, the definitive diagnosis of a herpesvirus infection requires either isolation of virus or detection of viral gene products.
For virus isolation, swabs of clinical specimens or other body fluids can be inoculated into susceptible cell lines and observed for the development of characteristic cytopathic effects.
This technique is most useful for the diagnosis of infection due to herpes simplex virus 1 and 2 or varicella-zoster virus because of their relatively short replicative cycles. The identification of cytomegalovirus by cell culture requires a longer period of time due to its prolonged period of replication.
Epstein-Barr virus does not induce cytopathic changes in cell culture systems and, therefore, can only be identified in culture by transformation of cord blood lymphocytes. Similarly, human herpes virus 6 and 7 have unique growth characteristics which make identification in cell culture systems difficult.
Newer and more rapid diagnostic techniques involve the detection of viral gene products. This can be done by applying fluorescence antibody directed against immediate-early or late gene products to tissue cultures after 24 to 72 hours of incubation. A positive result is the appearance of intranuclear fluorescence. A method which utilizes monoclonal antibodies to an immediate-early gene has been most useful for the identification of CMV.
Alternatively, fluorescence antibodies may be applied directly to cell monolayers or scrapings of clinical lesions, with intranuclear fluorescence again indicating a positive result. Recently developed diagnostic techniques that have clinical utility include in situ and dot-blot hybridization and, importantly, polymerase chain reaction DNA amplification.
This latter technique has proved most successful in the diagnosis of herpes simplex virus infections of the central nervous system, particularly when applied to cerebrospinal fluid. Importantly, this tool has been utilized to study the natural history of genital herpes simplex virus infections as well as identify new herpesvirus infections i. Kaposi's sarcoma herpesvirus. In addition to new tests for virus gene products and viral DNA, improved serologic assays are also becoming available, particularly the application of immunoblot technology to distinguishing herpes simplex virus 1 from 2 infections.
However, these tests are only useful for making a diagnosis in retrospect. Finally, the diagnosis of cytomegalovirus retinitis deserves special mention because it is made clinically by the presence of characteristic retinal changes. The diagnosis is further supported by the presence of cytomegalovirus viruria or viremia, but this is not an absolute requirement.
Of all the herpesviruses, herpes simplex virus type 1 and herpes simplex virus type 2 are the most closely related, with nearly 70 per cent genomic homology. These two viruses can be distinguished most reliably by DNA composition; however, differences in antigen expression and biologic properties also serve as methods for differentiation.
A critical factor for transmission of herpes simplex viruses, regardless of virus type, is the requirement for intimate contact between a person who is shedding virus and a susceptible host. After inoculation onto the skin or mucous membrane and an incubation period of four to six days, herpes simplex virus replicates in epithelial cells Figure As replication continues, cell lysis and local inflammation ensue, resulting in characteristic vesicles on an erythematous base.
Regional lymphatics and lymph nodes become involved: viremia and visceral dissemination may develop depending upon the immunologic competence of the host. In all hosts, the virus generally ascends the peripheral sensory nerves to reach the dorsal root ganglia. Replication of herpes simplex virus within neural tissue is followed by retrograde axonal spread of the virus back to other mucosal and skin surfaces via the peripheral sensory nerves. Virus replicates further in epithelial cells, reproducing the lesions of the initial infection, until infection is contained through both systemic and mucosal immunity.
Latency is established when herpes simplex virus reaches the dorsal root ganglia after anterograde transmission via sensory nerve pathways. In its latent form, intracellular herpes simplex virus DNA cannot be detected routinely unless specific molecular probes are utilized.
Mucocutaneous infections are the most common clinical manifestations of herpes simplex virus 1 and 2. Gingivostomatitis, which is usually caused by herpes simplex virus 1, occurs most frequently in children less than five years of age. Gingivostomatitis is characterized by fever, sore throat, pharyngeal edema and erythema, followed by the development of vesicular or ulcerative lesions on the oral and pharyngeal mucosa. Recurrent herpes simplex virus 1 infections of the oropharynx most frequently manifest as herpes simplex labialis cold sores , and usually appear on the vermillion border of the lip.
Intraoral lesions as a manifestation of recurrent disease are uncommon in the normal host but do occur frequently in immunocompromised individuals. Genital herpes is most frequently caused by herpes simplex virus 2 but an ever increasing number of cases are attributed to herpes simplex virus 1. Primary infection in women usually involves the vulva, vagina, and cervix Figure In men, initial infection is most often associated with lesions on the glans penis, prepuce or penile shaft.
In individuals of either sex, primary disease is associated with fever, malaise, anorexia, and bilateral inguinal adenopathy. Women frequently have dysuria and urinary retention due to urethral involvement. It is estimated that as many as 10 per cent of individuals will develop an aseptic meningitis with primary infection.
Sacral radiculomyelitis may occur in both men and women, resulting in neuralgias, urinary retention, or obstipation. The complete healing of primary infection may take several weeks. It has been recognized that the first episode of genital infection is less severe in individuals who have had previous herpes simplex virus infections at other sites, such as herpes simplex labialis.
Recurrent genital infections in either men or women can be particularly distressing. The frequency of recurrence varies significantly from one individual to another. It has been estimated that one-third of individuals with genital herpes have virtually no recurrences, one-third have approximately three recurrences per year, and another one-third greater than three per year.
Recent seroepidemiologic studies have found that between 25 percent and 65 percent of individuals in the United States in had antibodies to herpes simplex virus 2, and that seroprevalence is dependent upon the number of sexual partners.
If genital swabs from women with a history of recurrent genital herpes are subjected to polymerase chain reaction, virus DNA can be detected in the absence of culture proof of infection. This finding suggests the chronicity of genital herpes as opposed to a recurrent infection. Herpes simplex keratitis is usually caused by herpes simplex virus 1 and is accompanied by conjunctivitis in many cases.
It is considered the most common infectious cause of blindness in the United States. The characteristic lesions of herpes simplex keratoconjunctivitis are dendritic ulcers best detected by fluorescein staining. Deep stromal involvement has also been reported and may result in visual impairment. Herpes simplex virus infections can manifest at any skin site. Common among health care workers are lesions on abraded skin of the fingers, known as herpetic whitlows Figure Similarly, wrestlers, because of physical contact may develop disseminated cutaneous lesions known as herpes gladiatorum.
Neonatal herpes simplex virus infection is estimated to occur in approximately one in deliveries in the United States annually. Approximately 70 percent of the cases are caused by herpes simplex virus 2 and usually result from contact of the fetus with infected maternal genital secretions at the time of delivery.
Manifestations of neonatal herpes simplex virus infection can be divided into three categories: 1 skin, eye and mouth disease; 2 encephalitis; and 3 disseminated infection. As the name implies, skin, eye and mouth disease consists of cutaneous lesions and does not involve other organ systems Figure Involvement of the central nervous system may occur with encephalitis or disseminated infection, and generally results in a diffuse encephalitis.
The cerebrospinal fluid formula characteristically reveals an elevated protein and a mononuclear pleocytosis. Disseminated infection involves multiple organ systems and can produce disseminated intravascular coagulation, hemorrhagic pneumonitis, encephalitis, and cutaneous lesions. Diagnosis can be particularly difficult in the absence of skin lesions.
The mortality rate for each disease classification varies from zero for skin, eye and mouth disease to 15 per cent for encephalitis and 60 percent for neonates with disseminated infection. In addition to the high mortality associated with these infections, morbidity is significant in that children with encephalitis or disseminated disease develop normally in only approximately 40 per cent of cases, even with the administration of appropriate antiviral therapy.
Herpes simplex encephalitis is characterized by hemorrhagic necrosis of the inferiomedial portion of the temporal lobe Figure Disease begins unilaterally, then spreads to the contralateral temporal lobe. It is the most common cause of focal, sporadic encephalitis in the United States today, and occurs in approximately 1 in , individuals. Most cases are caused by herpes simplex virus 1. The actual pathogenesis of herpes simplex encephalitis requires further clarification, although it has been speculated that primary or recurrent virus can reach the temporal lobe by ascending neural pathways, such as the trigeminal tracts or the olfactory nerves.
Hemorrhagic necrosis of the temporal lobe due to HSV encephalitis. Clinical manifestations of herpes simplex encephalitis include headache, fever, altered consciousness, and abnormalities of speech and behavior. Focal seizures may also occur. The cerebrospinal fluid formula for these patients is variable, but usually consists of a pleocytosis with both polymorphonuclear leukocytes and monocytes present. The protein concentration is characteristically elevated and glucose is usually normal.
Historically, a definitive diagnosis could only be achieved by brain biopsy, since other pathogens may produce a clinically similar illness.
However, the application of polymerase chain reaction for detection of virus DNA has replaced brain biopsy as the standard for diagnosis. The mortality and morbidity are high, even when appropriate antiviral therapy is administered. At present, the mortality rate is approximately 30 per cent one year after treatment. In addition, approximately 70 per cent of survivors will have significant neurologic sequelae. Herpes simplex virus infections in the immunocompromised host are clinically more severe, may be progressive, and require more time for healing.
Manifestations of herpes simplex virus infections in this patient population include pneumonitis, esophagitis, hepatitis, colitis, and disseminated cutaneous disease. Individuals suffering from human immunodeficiency virus infection may have extensive perineal or orofacial ulcerations. Herpes simplex virus infections are also noted to be of increased severity in individuals who are burned. Transmission of herpes simplex virus is dependent upon intimate contact. Thus, herpes simplex virus 1 is usually transmitted by kissing or other contact with saliva, while herpes simplex virus 2 is usually a consequence of sexual contact.
Nosocomial spread of herpes simplex virus 2 has been documented, particularly in newborn intensive care units. Varicella-zoster virus is one of the most common viruses encountered by humans. Varicella-zoster virus is usually transmitted by airborne routes droplet spread with initial replication in the oropharynx Figure In the susceptible or seronegative individual, replication of virus in the oropharynx leads to primary viremia, with subsequent development of a vesicular rash.
The replication of varicella-zoster virus in vitro is similar to that for herpes simplex virus, although the period of replication is somewhat prolonged.
Varicella, or chickenpox, is the manifestation of primary varicella-zoster virus infection. This infection occurs most commonly in young children of preschool age and has a characteristic disseminated vesicular rash which appears after an incubation period of 14 to 17 days. The rash begins on the face and trunk and spreads to the extremities.
The lesions of chickenpox are initially vesicles which become pustular, crusted, and then scabbed prior to healing. The average duration of lesion formation is three to five days in the normal child; however, it is usually longer in adolescents and adults and certainly in the immunocompromised. At the time of primary infection, varicella-zoster virus may establish latency in dorsal root ganglia.
The recurrent form of varicella-zoster virus is herpes zoster or shingles. This form of infection, which is a reactivation of latent virus, typically manifests as a localized vesicular rash with a dermatomal distribution. The rash initially appears within the dermatome as erythema, which is soon followed by the development of vesicles Figure Some individuals will have coalescence of vesicles into bullous lesions.
New vesicles may form for five to seven days, then evolve through the sequence of healing described for the lesions of varicella. The average time to healing for individuals with shingles ranges from 10 to 21 days, depending upon the age and immune status of the individual.
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