|The Homepage of Dr. Edward K. Wagner|
Herpes simplex virus Research
HSV ReplicationUpon infecting a cell, HSV is immediately faced with an important "decision", whether to proceed to productive infection or whether to establish a latent infection. As outlined in the figure below, the most recent models posit that when viral DNA migrates to nuclear pods it is either circularized by cellular DNA repair enzymes acting on the "a" sequences or remains linear through the action of the immediate-early ICP0 protein, which inhibits cellular DNA repair. In the former case, latent infection ensues while in the latter, productive replication (vegetative) cycle of HSV gene expression, characterized by a progressive cascade of increasing complexity takes place.
The earliest genes expressed in the "immediate-early" or a phase are important in "priming" the cell for further viral gene expression and in mobilizing cellular transcriptional machinery. This phase is followed by the expression of a number of genes either directly or indirectly involved in viral genome replication--the "early" (b) phase; and finally, upon genome replication viral structural proteins are expressed in high abundance during the "late" (bg/g) phase.
The HSV replicative cycle is rapid compared to many other types of herpesviruses, but this general temporal classification fits the productive replication cycle of all, as well as smaller nuclear-replicating DNA viruses such as adenoviruses and papovaviruses.
Initial Steps in infection--virus entry
Virus entry requires sequential interaction between specific viral membrane glycoproteins and cellular receptors. Much of the recent work elucidating these receptors and the viral glycoproteins interacting with them has been carried out in the laboratories of P. Spear (Northwestern University), and G. Cohen and R. Eisenberg of the University of Pennsylvania.
Upon entry the nucleocapsid is transported to the nuclear pores, where viral DNA is released into the nucleus. The viral genome is accompanied by the a-TIF protein which functions in enhancing immediate early viral transcription via cellular transcription factors. The virion-associated host shutoff protein (vhs--UL41) appears to remain in the cytoplasm where it causes the disaggregation of polyribosomes and degradation of cellular and viral RNA.
The HSV-1 transcription program
Immediate early gene expressionFive HSV genes: a4--ICP4, a0--ICP0, a27--ICP27/UL 54, a22--ICP22/US1, and a47--ICP47/US12 are expressed and function the earliest stages of the productive infection cycle. This stage of infection is termed the "immediate-early" or "a" phase of gene expression, and is mediated by the action of a-TIF through its interaction with cellular transcription factors at specific enhancer elements associated with the individual a-transcript promoters.
Proteins encoded by the a4, a0, and a27 transcripts act to activate viral gene expression at the level of transcription, or at least, mRNA expression. They functionally interact to form nuclear complexes with viral genomes. Surprisingly, only two (a4 and a27) have extensive areas of sequence similarity among a large number of alpha-herpesviruses, and only amino acid sequences in a27 appear to be extensively conserved among the more distantly related beta- and gamma-herpesviruses. Both of the two other a proteins, a22 and a47 are dispensable for virus replication in many types of cultured cells, but a22 is required for HSV replication in others and may have a role in maintaining the virus's ability to replicate in a broad range of cells in the host -- perhaps by providing some types of cells with the capacity to express of a group of late transcripts. The a47 protein appears to have a role in modulating host response to infection by specifically interfering with the presentation of viral antigens on the surface of infected cells.
Early gene expressionActivation of the host cell transcriptional machinery by the action of a gene products, results in the expression of the early or b genes. The promoters for such genes (exemplified by the thymidine kinase transcript promoter) have served as models for "typical" eukaryotic promoters.
Seven of these are necessary and sufficient for viral DNA replication under all conditions: DNA polymerase (UL30), DNA binding proteins (UL42 and UL29 or ICP8), ORI binding protein (UL9), and the helicase/primase complex (UL5, 8, and 52). When sufficient levels of these proteins have accumulated within the infected cell, viral DNA replication ensues. Other early proteins are involved in increasing the deoxyribonucleotide pools of the infected cells, while still others appear to function as repair enzymes for the newly synthesized viral genomes. These accessory proteins are "non-essential" for virus replication in that cellular products can substitute for their function in one or another cell type or upon replication of previously quiescent cells; however, disruptions of such genes often have a profound effect upon viral pathogenesis, and/or ability to replicate in specific cells.
Genome replication and late gene expression
The vegetative replication of viral DNA represents a critical and central event in the viral replication cycle. High levels of DNA replication irreversibly commit a cell to producing virus, which eventually results in cell destruction. DNA replication also has a significant influence on viral gene expression. Early expression is significantly reduced or shut off following the start of DNA replication, while late genes begin to be expressed at high levels.
Transcripts expressing late genes can be divided into two subclasses: "leaky-late" (bg) and "strict" late (g). Promoters controlling expression of both classes are similar in that both have elements near the transcription start site (cap site) which are required for promoter activity, but the location of other elements can differ. The bg transcripts are expressed at low levels prior to DNA replication, but reach maximum expression after viral DNA replication has been initiated. In contrast, g transcripts are difficult to detect at all until the onset of viral DNA replication.
Immunofluorescence studies show that DNA replication and late transcription occurs at discrete sites, or "replication compartments"in the nucleus. Prior to DNA replication, the a4 protein and the b single stranded DNA binding protein ICP8 (UL29) are distributed diffusely throughout the nucleus-as are cellular transcription complexes. Concomitant with viral DNA replication, the distribution of these proteins changes to a punctate pattern.This change involves interaction with a0 and a27.
DNA microarray analysis of viral transcript abundance at various times after infection
More than 30 HSV-1 gene products are structural components of the virion and all are expressed with late kinetics. Capsid assembly, enveloping, and is a complex process. Insight into the general outline of the process were established by careful electronmicroscopic studies by W. Gibson along with the biochemical analyses by P. Desai and S. Person all at Johns Hopkins University and work by Frazer Rixon at the MRC Virology Unit in Glasgow. A great impetus to the understanding of the biochemical steps in viral morphogenesis was provided by the work of Homa and colleagues working in the research labs of the Pharmacia and Upjohn Corporations. They fused recombinant baculovirus vectors to express all the HSV proteins required for capsid assembly in insect cells, and demonstrated full capsid morphogenesis--they and others have later done this in a cell free environment. HSV capsids assemble around viral scaffolding proteins in the nucleus, and then other viral proteins interact with replicated viral DNA to allow DNA encapsidation. The encapsidated DNA is not associated with histones, but highly basic polyamines (perhaps synthesized with viral enzymes) appear to facilitate the encapsidation process and full capsids presumably associate with tegument (matrix) proteins near the nuclear membrane.
Virus Envelopement and ReleaseThe envelopement of alpha herpesviruses is a complex process, and work by D. Johnson (Oregon Health Sciences University, C. Grose (Univ. of Iowa), Tony Minson (Cambridge University) have provided useful insights into the process. Recently, Mettenleiter and colleagues have provided persuasive evidence that viral membrane formation is by a double envelopment process. Mature capsids bud through the inner nuclear membrane that contains viral glycoproteins. In the early maturation process in the nucleus, capsids appear to be surrounded by the primary tegument protein, UL31 and this directs the budding through the inner nuclear membrane into which the UL31 and UL34 phosphorylated membrane protein has been inserted. These primarily enveloped capsids then bud through the outer nuclear membrane where the primary envelope is lost. The cytoplasmic capsids then associate with the numerous tegument proteins of the mature virion, including a-TIF and vhs, which appear to functionally interact to help final envelopment. Final envelopment takes place as the mature capsids and associated tegument proteins bud into exocytotic vesicles, the membranes of which contain all the glycoproteins associated with the mature virions. Infectious virions can either remain cell associated within these vesicles, and spread to uninfected cells via virus-induced fusion, or can be released from the cell in exocytotic vesicles.