Saturday, March 25, 2023
This activity is intended for scientists, physicians, and other allied health professionals working in the fields of HIV, hematology, oncology, immunology, infectious diseases, virology, and molecular & cellular biology.
The goal of this activity is to enhance the quality of clinical practice by healthcare professionals involved in the care of individuals with HIV/AIDS by reporting state-of-the-art treatment approaches and clinical strategies for the management of HIV/AIDS.
Upon completion of this activity, participants should be able to:
The National Institutes of Health/Foundation for Advanced Education in the Sciences (NIH/FAES) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.
The NIH/FAES designates this educational activity for a maximum of 1 hour in category 1 credit towards the AMA Physician's Recognition Award. Each physician should claim only those hours of credit that he/she actually spent in the educational activity.
For questions regarding the content of this activity, contact the accredited provider for this CME/CE activity noted above. For technical assistance, contact [email protected]
There are no fees for participating in or receiving credit for this online educational activity. For information on applicability
and acceptance of continuing education credit for this activity, please consult your professional licensing board.
This activity is designed to be completed within the time designated on the title page; physicians should claim only those
credits that reflect the time actually spent in the activity. To successfully earn credit, participants must complete the
activity online during the valid credit period that is noted on the title page.
Follow these steps to earn CME/CE credit:
You may now view or print the certificate from your CME/CE Tracker. You may print the certificate but you cannot alter it.
Credits will be tallied in your CME/CE Tracker and archived for 5 years; at any point within this time period you can print
out the tally as well as the certificates by accessing "Edit Your Profile" at the top of your Medscape homepage.
The credit that you receive is based on your user profile.
processing....
The session on virus replication and immune responses to infection included presentations on human papillomavirus (HPV), Epstein-Barr Virus (EBV), human herpesvirus 8 (HHV-8), and the simian Kaposi's sarcoma (KS)-like viruses retroperitoneal fibromatosis herpesvirus (RFHV) and rhesus monkey rhadinovirus (RRV). In addition, new information was presented on the role of hypoxia in lytic replication of HHV-8 and EBV.
HHV-8 has been detected in a number of different human and animal cell types, including endothelial and epithelial cells, B cells, keratinocytes, and macrophages. Akula and colleagues[1] at the University of Kansas Medical Center, Kansas City, presented data showing that this broad cellular tropism was in part due to the interaction of the HHV-8 envelope glycoproteins gB and gpK8.1 with the ubiquitous host cell surface molecule heparan sulfate. Since heparan sulfate is found in association with many cell types, the investigators searched for additional cellular molecules that may act as coreceptors for HHV-8. Sequencing data revealed that the HHV-8 gB open reading frame contains the minimal amino acid sequence RGD required to bind cell surface integrins. This group's data suggest that the alpha3beta1 integrin is a cellular receptor utilized by HHV-8 that facilitates virus particle entry into the host cell. All of the cell types with detectable HHV-8 express alpha3beta1 integrin. Antibodies to alpha3beta1 integrin block HHV-8 infection in human microvascular endothelial cells. Immunoprecipitation experiments showed that gB expression was required for binding to alpha3beta1 integrin. Akula and colleagues then showed that the interaction of gB with alpha3beta1 activates focal adhesion kinase (FAK) by phosphorylation and sets up a signal transduction cascade, which induces actin polymerization of microfilaments and may assist HHV-8 particle entry by endocytosis (Figure 1).
Figure 1. A model of HHV-8 interactions with target cells and mechanism of entry.
Garrigues and Rose[2] from the University of Washington, Seattle, presented information on the gB homologue in retroperitoneal fibromatosis herpesvirus (RFHV), a lymphoproliferative disease of rhesus macaques that is similar to AIDS-related KS. They also identified a conserved N-terminal RGD motif when the HHV-8 gB sequence was compared with the RFHV sequence. Using antibody-blocking studies, they found that gB RGD peptide binds preferentially to the "vitronectin" alphaVbeta3 cellular integrin receptor, despite the presence of more highly expressed integrins on the fibrosarcoma and dermal microvascular cell lines tested.
An investigation of the attachment of HHV-8 to different cell types, using flow cytometry, was undertaken by Dezube and colleagues[3] at Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. Microvascular dermal endothelial cells (MVDEC) had the best attachment of HHV-8, followed by B cells and monocytes. T cells were able to bind HHV-8 at a low level, and natural killer cells had no detectable binding. The investigators then examined HHV-8 infection in the MVDEC by electron microscopy and found virions in cytoplasmic vesicles and free in the cytosol. An examination of the viral genomes during initial infection of MVDEC found that linear and circular genomes appeared 24 hours postinfection. When RNA transcripts from these infected MVDEC cells were examined, the latently expressed ORF 74 (G-protein coupled receptor) mRNA levels fluctuated in cyclic variations every 48-72 hours, while the lytic genes T1.1 and K8.1 were continuously detectable after 6 days in culture, indicating a mixed lytic and latent infection.
Lin and Yuan[4] from the University of Pennsylvania, Philadelphia, described the identification of the HHV-8 lytic origin of replication (ori-Lyt). Herpesviruses use host cell enzymes for replication when they are in latency. However, when induced into lytic replication, virus-encoded replication enzymes are required. In addition to ori-Lyt, an origin binding protein (OBP) is required. Lin and Yuan identified 2 regions located between the K4.2 -K5 and K12-ORF 71 genes that might function as an origin of lytic replication. Mapping analysis revealed a 1.7-kb DNA sequence that is the core domain for ori-Lyt. The K8 gene of HHV-8 was found to bind to this region using the chromatin immunoprecipitation (Chip) method. Of interest, the HHV-8 K8 gene is similar in size and genome location and has some sequence homology with the BZLF1 gene of EBV. The BZLF1 gene product, Zta, is known to be the OBP of EBV. This suggests that the K8 protein may function as the HHV-8 OBP.
Wu and coworkers[5] at the Johns Hopkins Hospital and School of Medicine, Baltimore, Maryland, reported on the mechanism of cell cycle arrest in HHV-8-infected cell lines. The K8 gene of HHV-8 encodes replication-associated protein (Rap). These researchers showed that Rap accumulates in HHV-8 replication compartments 12-24 hours into lytic cycle replication and its expression coincides with an increased expression of cellular C/EBP-alpha and p21 proteins. C/EBP-alpha and p21 proteins are important in cell differentiation and block cell cycle progression at G1/S. These data suggest that Rap must associate with C/EBP-alpha and p21 in order to block the cell cycle in primary effusion lymphoma cells.
Talanin and associates[6] at the National Cancer Institute (NCI), Bethesda, Maryland, and George Washington University, Washington, DC, presented data on histone deacetylase inhibitors (HDAI) that might be useful clinically to reactivate HHV-8 from latent reservoirs. These inhibitors induce transcription of cellular genes involved in tumor suppression, apoptosis, and maturation -- genes that are often "silenced" in virus-transformed cells and tumors. All of the HDAI that were tested induced lytic cycle replication in BC3 and BCBL1 cells, as measured by flow cytometry expression of PF8, mRNA expression of K8.1, ORF 50, and ORF 59, and virion production measured by electron microscopy. These data suggest that HDAI may be useful clinically to treat KS by reactivating virus, leaving the cells susceptible to apoptosis and the virus to antiherpesvirus agents.
DeWire and Damania[7] from the University of North Carolina at Chapel Hill presented data on early lytic gene expression of another KS-like virus detected in monkeys, rhesus monkey rhadinovirus (RRV) (Figure 2). Data were presented on a polycistronic transcript encoding the ORF 50/rta transactivator, R8 and R8.1. They found that despite only 55% gene sequence homology, RRV ORF 50 could substitute in the HHV-8 genome and transactivate HHV-8 promoters. Also, the ORF 50 gene of HHV-8 can substitute for RRV ORF 50. These 3 RRV genes are structural homologues of HHV-8 and suggest conservation of gene function between HHV-8 and RRV, validating the use of RRV as an animal model of KS.
Figure 2. The herpesvirus tree, illustrating the lineage of the primate HHV-8/KSHV-like herpesviruses.
Haque and colleagues[8] at the NCI presented data on the hypoxia response elements (HRE) in the HHV-8 genome. Hypoxia induces production of hypoxia-inducible factors (HIFs), transcription factors that activate gene expression of cellular genes in response to hypoxic conditions. The cellular genes affected by hypoxia include genes involved in energy metabolism, angiogenesis, erythropoiesis, apoptosis, and proliferation. A recent paper by this group[9] showed that hypoxia could increase lytic replication of HHV-8. Three putative HRE elements have been identified in the ORF 34 gene of HHV-8 by sequence analysis of the computer database. One of these HREs located upstream of the ORF 34 gene could be induced 10-fold in HEP 3B cells transfected with HHV-8 genes and grown in hypoxia. HIF-2 alpha was found to induce the ORF 34 gene. Preliminary data have also shown that the rta gene (ORF 50) can be moderately upregulated by hypoxia.
Additional information on the effect of hypoxia on EBV lytic replication was presented as a poster.[10] Increases in the expression of the EBV capsid antigen GP125 and the ZTA or ZEBRA protein were observed when cells were cultured under hypoxic conditions (1% O2). In addition, mRNA transcripts of the BZFL1 gene and EBNA1 were increased when EBV cell lines were grown in 1% O2.
In the second plenary lecture of the virus replication session, Dr. Hutt-Fletcher explored the interactions between EBV envelope glycoproteins and their cellular receptors on epithelial and B cells during attachment and fusion.[11] The 2 human cell types most susceptible to productive infection by EBV are B cells and epithelial cells. The EBV virion proteins that mediate binding and fusion to the host cell membrane differ in B cells and epithelial cells. Initial attachment of an EBV particle occurs through binding of the viral gp220/350 protein to the CD21 receptor on host B cells. Three additional viral envelope glycoproteins -- gH, gL, and gp42 -- are required for fusion onto the host B cell. It has been found that the direct interaction requires gp42 expression, which binds to the MHC-Class II antigen, the coreceptor for EBV. However when EBV-host cell interactions were examined in epithelial cells, it was discovered that the addition of exogenous soluble gp42 blocked attachment of EBV. The EBV coreceptor on epithelial cells is presently unknown. A model of infection of B cells and epithelial cells is shown in Figure 3. EBV can express 2-part or 3-part protein complexes. When EBV particles are available to infect B cells, the 3-part complex containing gH, gL, and gp42 is present on the virus particle. During lytic replication in B cells, gp42 is downregulated, and the virus particles released lack gp42. These nascent EBV particles are then able to selectively infect epithelial cells. When epithelial cells undergo lytic replication the gp42 gene is then expressed on the virus and is available to interact with the MHC-Class II antigen on uninfected B cells. This suggests that gp42 is a molecular switch that allows EBV to infect both B cells and epithelial cells.
Figure 3. Proposed model for explaining the change in expression of the EBV glycoprotein gp42. During EBV replication in B cells, gp42 is downregulated, allowing EBV to infect epithelial cells. When epithelial cells undergo lytic replication, gp42 expression is upregulated, so EBV can efficiently infect B cells.
Ruvolo and colleagues[12] discussed the roles of the EBV-encoded SM protein on host cell gene expression. SM protein, also known as Mta or EB2, is a delayed intermediate-early EBV gene product that functions as a posttranscriptional gene regulator. Previous work has shown that SM protein shuttles mRNA transcripts from the nucleus to the cytoplasm, inhibits expression of genes containing introns, and activates genes without introns.[13] Microarray analysis was performed to access the full range of activities of the SM protein on host cell gene expression in B cells. Most cellular genes except for the interferon-stimulated genes (ISGs) were inhibited. ISGs are induced by type-1 interferons that have an interferon-stimulated response element (ISRE) in the promoter region. However, SM activity did not lead to an increase in interferon secretion, but led to an increase of STAT1 (signal transducers and activators of transcription) mRNA transcripts and altered the STAT1 alpha/beta ratio. STAT1 is part of the ISRE that mediates transcription of ISG. Induction of interferon-stimulated genes led to a decrease in cell proliferation, but did not lead to cell-cycle arrest or apoptosis.
Dr. Laimins[14] from the Department of Microbiology and Immunology at Northwestern University, Chicago, Illinois, gave a plenary lecture on human papillomavirus (HPV) replication in epithelial cells. HPV-31 infection is causally linked with cervical carcinoma and is classified as a high-risk papillomavirus infection. Initial infection is thought to occur in epithelial stem cells or transit -amplifying cells located in the lower levels of the stratified epithelium.[14,15] HPV-31 infection results in an altered pattern of differentiation, as illustrated in Figure 4. During differentiation, normal cells lose their nuclei while virus-infected cells retain their nuclei. In addition, the synthesis of HPV viral DNA and expression of late viral genes increases as the cells differentiate. To investigate the change from maintenance of the episomal HPV at about 50 copies/cell early in the differentiation process to the massive production of virus particles, a tissue culture method for examining terminal differentiation in HPV-infected keratinocytes was developed. E1 and E2 are HPV viral proteins required for virus replication.[16] By studying the replication process, Laimins[14] has found that during stable maintenance of the HPV genome in basal cells, the E1 protein is transcribed from the p97 promoter. The E2 protein regulates the levels of E1. When the basal cells differentiate to suprabasal cells, a promoter shift occurs and E1 and E2 are both transcribed at high levels. This leads to a high level of HPV replication as shown in Figure 5.
Figure 4. HPV infection induces altered patterns of differentiation and changes in viral gene replication during host cell differentiation.
Figure 5. A promoter shift occurs when basal cells differentiate to suprabasal cells, leading to high-level transcription of E1 and E2 from the p742 promoter and a high rate of HPV replication.
