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Participatory sequences in the herpes simplex virus thymidine kinase (HSV tk) recoding are determined in a herpesvirus mutant. The findings demonstrate a novel mechanism that relies on the capacity of the G-rich sequences to generate unique inter- or intramolecular structures. Specifically, the sole influence of G-strings to activate a net +1 recoding in cultured cells is determined. On the other hand, mediation of recoding by the G-string structure inside the ribosome is proposed to proceed either by the Hoogsteen or non-Watson-Crick interactions.

Abstract: Mammalian alphaherpesviruses are major causes of human and veterinary disease. During productive infection, these viruses exhibit complex and robust patterns of gene expression. These viruses also form latent infections in neurons of sensory ganglia in which productive cycle gene expression is highly repressed. Both modes of infection provide advantageous opportunities for regulation by microRNAs. Thus far, published data regarding microRNAs are available for six mammalian alphaherpesviruses. No microRNAs have yet been detected from varicella zoster virus. The five other viruses—herpes simplex viruses-1 and -2, herpes B virus, bovine herpesvirus-1, and pseudorabies virus—representing both genera of mammalian alphaherpesviruses have been shown to express microRNAs. In this article, we discuss these microRNAs in terms of where they are encoded in the viral genome relative to other viral transcripts; whether they are expressed during productive or latent infection; their potential targets; what little is known about their actual targets and functions during viral infection; and what little is known about the interactions of these viruses with the host microRNA machinery. This article is part of a Special Issue entitled: “MicroRNAs in viral gene regulation”. [Copyright &y& Elsevier]

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter. • HCMV infection induces SAMHD1 expression and phosphorylation • SAMHD1 restricts HCMV gene expression before virus replication • SAMHD1 deficiency limits HCMV entry into the quiescent stage of infection • HCMV restriction by SAMHD1 is mediated by limiting NF-κB activation SAMHD1 has been identified as a cellular antiviral restriction factor. Kim et al. report that HCMV is restricted by SAMHD1 through inhibition of viral gene expression. They show that depletion of SAMHD1 increases activation of the NF-κB immune pathway, which paradoxically increases gene expression from the immediate-early viral promoter. [ABSTRACT FROM AUTHOR]

Herpesviruses, like most DNA viruses, replicate and package their genomes into capsids in the host cell nucleus. Capsids then transit to the cytoplasm in a fascinating process called nuclear egress, which includes several unusual steps: Movement of capsids from the nuclear interior to the periphery, disruption of the nuclear lamina, capsid budding through the inner nuclear membrane, and fusion of enveloped particles with the outer nuclear membrane. Here, we review recent advances and emerging questions relating to herpesvirus nuclear egress, emphasizing controversies regarding mechanisms for capsid trafficking to the nuclear periphery, and implications of recent structures of the two-subunit, viral nuclear egress complex for the process, particularly at the step of budding through the inner nuclear membrane. [ABSTRACT FROM AUTHOR]

Summary: Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the “circle.” The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a “hybrid” of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both. [Copyright &y& Elsevier]

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the ‘connector loop’ of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic ‘plug’ from the UL54 peptide and Ile135 of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44. [ABSTRACT FROM AUTHOR]

The most direct approach to elucidating the roles of herpes simplex virus (HSV) proteins in the viral replicative cycle has been to isolate temperature-sensitive, cytolysis-resistant, and drug-resistant mutants that exhibit alterations in the synthesis or activity of these proteins. The development of procedures for the introduction of temperature-sensitive mutations into physically defined regions of the viral genome and for fine mapping of these mutations has proven especially valuable. Thus, (1) hydroxylamine mutagenesis of the HSV-1 BglII I fragment (coordinates 0.312-0.415) has facilitated the genetic and functional characterization of the gene for the major viral DNA-binding protein of 130 K molecular weight; (2) the selection of a mutant conditionally able to render infected cells resistant to immune cytolysis has led to identification of an HSV gene involved in the processing of viral glycoproteins; and (3) the combined use of temperature-sensitive and drug-resistant mutants has led to a better definition of the physical limits and functional domains of the gene for HSV DNA polymerase. [ABSTRACT FROM AUTHOR]