Expanding the Menu of HIV Prevention Options: A Qualitative Study of Experiences with Long-Acting Injectable Cabotegravir as PrEP in the Context of a Phase II Trial in the United States.

Adherence challenges with oral pre-exposure prophylaxis have stimulated interest in alternate modes of administration including long-acting injections. We conducted 30 in-depth interviews with 26 male trial participants and 4 clinical providers in a Phase IIa study (ÉCLAIR) evaluating the use of long-acting cabotegravir (CAB-LA) injections in New York and San Francisco. Interviews exploring attitudes and experiences with CAB-LA were audiotaped, transcribed, and analyzed using thematic content analysis. Despite a high frequency of some level of side effects, almost all participants reported being interested in continuing with CAB-LA, versus a daily oral, due to its convenience and the perceived advantage of not worrying about adhering to pills. Providers reinforced the importance of CAB-LA as a prevention option and the need for guidelines to assist patient decision-making. Further research is needed on the acceptability of CAB-LA among men and women at higher risk for HIV in different settings.

The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation.

Because of the heterogeneity of chromatin, the site of integration of human immunodeficiency virus (HIV) in the genome could have dramatic effects on its transcriptional activity. We have used an HIV-1-derived retroviral vector, in which the green fluorescent protein is under the control of the HIV promoter, to generate by infection 34 Jurkat clonal cell lines each containing a single integration of the HIV-1 vector. In the absence of Tat, a 75-fold difference in expression level between the highest and lowest expressing clones was observed. Basal promoter activity was low in 80% of the clones and moderate to high in the remaining 20% of clones. We found that differences in expression levels are due to the integration site and are not controlled by DNA methylation or histone acetylation. Tat activated transcription in each clone, and an inverse correlation was observed between basal transcriptional activity and inducibility by Tat. These observations demonstrate that the chromatin environment influences basal HIV gene expression and that the HIV Tat protein activates transcription independently of the chromatin environment.

Gene activation by Varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Rc of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1.

Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties.

Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type 1: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat- or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA.

Lessons to be learned from varicella-zoster virus.

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells.

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins.

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses. The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression. In transfection assays, IE4 has been shown to enhance activation induced by IE62. To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells. In single transfections, IE4 was predominantly found in cytoplasm. In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining. This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution. The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required. IE62 remained nuclear whether IE4 was present or not. These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues. In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei. This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62.

Varicella-zoster virus gene regulation.

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter.

Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63.

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.

Characterization of the regulatory functions of varicella-zoster virus open reading frame 4 gene product.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a protein with a predicted molecular weight of 51,540 presenting amino acid sequence homology with the immediate-early regulatory protein ICP27 of herpes simplex virus type 1. To investigate the regulatory properties of the ORF4 gene product, we performed a series of transient expression assays in Vero cells, using a plasmid expressing ORF4 as effector and several VZV genes and heterologous genes as targets. The VZV target plasmids contained promoter/regulatory regions from genes belonging to the three putative VZV kinetic classes fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target plasmids consisted of promoter/regulatory regions of human cytomegalovirus, Rous sarcoma virus, and human immunodeficiency virus type 1 fused to the reporter gene. These experiments demonstrated that the ORF4 gene product activated expression of ORF62 in a dose-dependent fashion but had no effect on the expression of the three other putative immediate-early genes (ORF4, ORF61, and ORF63). When various amounts of ORF4 were transfected in the presence of early gene promoters, dose-dependent transactivation was evidenced with the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters; interestingly, little activity was detected with the promoter of the DNA polymerase gene (ORF28). No activation of late gene expression, represented by the glycoprotein I and glycoprotein II genes, was seen even over a wide range of concentrations of input ORF4 plasmid. Expression of pCMVCAT, pRSVCAT, and pHIVCAT was also stimulated by the ORF4 gene product. CAT mRNA analysis showed that activation of VZV target promoters occurs at the transcriptional and/or posttranscriptional level.

Characterization of regulatory functions of the varicella-zoster virus gene 63-encoded protein.

Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression experiments strongly suggest that the VZV gene 63 protein could play a pivotal role in the repression of IE gene expression as well as in the activation of early gene expression.