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Abstract: Some of the most successful antiviral agents currently available are effective against herpes simplex virus. However, resistance to these drugs is frequently associated with significant morbidity, particularly in immunocompromised patients. In addition to the clinical implications of drug resistance, the range of biological processes exploited by the virus to attain resistance while maintaining pathogenicity is proving to be surprising. These mechanisms, which include ribosomal frameshifting, induced infidelity of the DNA polymerase, and internal ribosome entry, are discussed. [Copyright &y& Elsevier]

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]

We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion it the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-elF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single- base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs. [ABSTRACT FROM AUTHOR]

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice. [ABSTRACT FROM AUTHOR]

A double-guanine-insertion mutation within a run of guanines in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. This mutation was engineered into strain KOS, and stocks were generated from single plaques. Plaque autoradiography revealed that most plaques in such stocks exhibited low levels of TK activity, while ∼3% of plaques exhibited high levels of TK activity, indicating a remarkably high frequency of phenotypic reversion. This virus was able to reactivate from latency in mouse ganglia; a fraction of the reactivating virus expressed a high level of TK activity due to an additional G insertion, suggesting that the observed genetic instability contributed to pathogenicity. [ABSTRACT FROM AUTHOR]

A collection of genomic DNA sequences of herpes simplex virus (HSV) strains has been defined and analyzed, and some information is available about genomic stability upon limited passage of viruses in culture. The nature of genomic change upon extensive laboratory passage remains to be determined. In this report we review the history of the HSV-1 KOS laboratory strain and the related KOS1.1 laboratory sub-strain, also called KOS (M), and determine the complete genomic sequence of an early passage stock of the KOS laboratory sub-strain and a laboratory stock of the KOS1.1 sub-strain. The genomes of the two sub-strains are highly similar with only five coding changes, 20 non-coding changes, and about twenty non-ORF sequence changes. The coding changes could potentially explain the KOS1.1 phenotypic properties of increased replication at high temperature and reduced neuroinvasiveness. The study also provides sequence markers to define the provenance of specific laboratory KOS virus stocks. [ABSTRACT FROM AUTHOR]

UL24 is conserved among all Herpesviridae . In herpes simplex virus 1 (HSV-1), UL24 mutations lead to reduced viral titers both in cell culture and in vivo , and reduced pathogenicity. The human cytomegalovirus ortholog of UL24 has a gene regulatory function; however, it is not known whether other UL24 orthologs also affect gene expression. We discovered that in co-transfection experiments, expression of UL24 correlated with a reduction in the expression of several viral proteins and transcripts. Substitution mutations targeting conserved residues in UL24 impaired this function. Reduced transcript levels did not appear attributable to changes in mRNA stability. The UL24 ortholog of Herpes B virus exhibited a similar activity. An HSV-1 mutant that does not express UL24 produced more viral R1 and R2 transcripts than the wild type or rescue virus relative to the amount of viral DNA. These results reveal a new role for HSV-1 UL24 in regulating viral mRNA accumulation. [ABSTRACT FROM AUTHOR]

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with ~80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs. [ABSTRACT FROM AUTHOR]

To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRa), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRa did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections. [ABSTRACT FROM AUTHOR]

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia. [ABSTRACT FROM AUTHOR]