Transcription Of Viral Immediate-early Genes Research Articles

The Role of VP16 in the Life Cycle of Alphaherpesviruses.

The protein encoded by the UL48 gene of alphaherpesviruses is named VP16 or alpha-gene-transactivating factor (α-TIF). In the early stage of viral replication, VP16 is an important transactivator that can activate the transcription of viral immediate-early genes, and in the late stage of viral replication, VP16, as a tegument, is involved in viral assembly. This review will explain the mechanism of VP16 acting as α-TIF to activate the transcription of viral immediate-early genes, its role in the transition from viral latency to reactivation, and its effects on viral assembly and maturation. In addition, this review also provides new insights for further research on the life cycle of alphaherpesviruses and the role of VP16 in the viral life cycle.

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human Alphaherpesviruses.

Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.

A single amino acid substitution in herpes simplex virus type 1 VP16 inhibits binding to the virion host shutoff protein and is incompatible with virus growth.

In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAdeltaSma showed that VP16(L344A) was able to complement the growth of 8MAdeltaSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

Nucleotide and deduced amino acid sequences of the gene encoding virion protein 16 of herpes simplex virus type 2

A virion protein (VP16) of herpes simplex virus (HSV) activates transcription of viral immediate-early genes. We have determined the nucleotide sequence of the virion activator gene from HSV-2, which we term VP16-2. Comparison of the deduced amino acid sequences of the HSV-1 and HSV-2 homologs shows that the VP16-2 gene product retains features determined to be important for transcriptional activation by VP16.

Codons 262 to 490 from the herpes simplex virus ICP4 gene are sufficient to encode a sequence-specific DNA binding protein

The HSV-1 immediate early (IE) protein ICP4 (alpha 4, IE175, Vmw175) is an oligomeric molecule which activates transcription of viral early genes, represses transcription of viral IE genes, and binds to specific sequences in certain viral promoters. The extent to which these functions are interrelated has not been fully established. We have expressed truncated portions of the ICP4 gene in E. coli as trpE fusion proteins. DNA-binding studies with these hybrid proteins revealed that ICP4 residues 262 to 490 are sufficient for sequence-specific DNA-binding. DNA-binding was not detected with polypeptides extending from residue 262 to 464 or from residue 306 to 490. Multiple bands of protein-DNA complexes observed in gel mobility shift assays indicate that residues 262 to 490 may also contribute to the oligomerization of ICP4.

HSV-1 DNA sequence determining intraperitoneal pathogenicity in mice is required for transcription of viral immediate-early genes in macrophages.

The relationship between intraperitoneal (ip) pathogenicity in vivo of herpes simplex virus type 1 (HSV-1) and infection of macrophages (m phi) in vitro was studied. The apathogenic HSV-1 strain HFEM disappeared from the peritoneum of infected mice following ip inoculation, while the pathogenic F strain persisted in the peritoneum and penetrated the mouse nervous system, and eventually the mice died, showing severe neurological signs. When peritoneal m phi were infected in vitro, a direct correlation with pathogenicity in vivo was found with several HSV-1 strains and recombinants. HSV-1 strains (F, KOS, R-M1C1) which were pathogenic for mice by the ip route, induced cytopathic effect (CPE) in m phi in vitro. Strain F transcribed viral immediate-early genes and synthesized viral DNA in m phi that were treated with L-cell conditioned medium (as a source of colony-stimulating factor) prior to infection. Apathogenic HSV-1 strains (HFEM, R-15, R-19) did not cause CPE in m phi. The HFEM strain was already blocked in the transcription of viral alpha genes in the infected m phi, but replicated well in control BSC-1 cells. An intratypic recombinant (R-M1C1), produced by cotransfection of HFEM DNA with a cloned Mlul-Mlul DNA fragment (coordinates 0.7615-0.796) from HSV-1 strain F, that was shown [Becker et al. (1986). Virology 149, 255-259] to have regained partial ip virulence for mice, now transcribed alpha genes, synthesized viral DNA, and induced CPE in m phi. It appears that the viral DNA fragment responsible for ip virulence is involved in tissue-specific recognition of virus by infected m phi, a function necessary for transcription of viral alpha genes.