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Activation of Human Immunodeficiency Virus by Herpes Simplex Virus Marjorie P. Golden, Sunyoung Kim, Scott M. Hammer, Elizabeth A. Ladd, Priscilla A. Schaffer, Neal DeLuca, and Mary A. Albrecht
Division of Infectious Diseases, New England Deaconess Hospital, Laboratory ofTumor Virus Genetics, Dana Farber Cancer Institute, and Harvard Medical School, Boston, Massachusetts
Human immunodeficiency virus (HIV) type 1 is well established as the cause of AIDS [1-3]. In adults infected with HIV-1, clinical immunodeficiency appears after a mean incubation period of 8-10 years [4]. This prolonged clinical latency has led to speculation that, in certain individuals, infectious and other cofactors might affect disease progression. Up-regulation ofHIV expression in vitro is mediated by cytokines, protein kinase C activators, and heterologous viruses [5-10]. Antigens of several herpcsviruses, specifically cytomegalovirus (CMV) and Epstein-Barr virus, stimulate macrophages to produce cytokines, which in turn can up-regulate HIV expression in persistently infected monocytic and T lymphocytic cell lines [5]. The ability of heterologous DNA viruses to activate HIV has been studied most extensively using a chimeric plasmid containing the HIV long terminal repeat (LTR) linked to chloramphenicol acetyltransferase (CAT) as a reporter gene. When cells were transfected with this construct, CAT expression was increased after infection with herpes simplex virus (HSV) types 1 and 2, CMV, or varicella-zoster virus (VZV) [10]. In a transient cotransfection system, the HSV immediate-early (IE) regulatory proteins ICPOand ICP4, but
Received 30 January 1992; revised 13 April 1992. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, September 1991 (abstract 504). Financial support: National Institutes of Health (AI-OI0 15 to M.A., AI30897 to S.K., HL-4351 0 and -42112 to S.H .. AI-27431 to N.D., and CA20260 to P.S.); Department of Defense (DAMD 17-90-C-0106 to S.H.). Reprints or correspondence: Dr. Mary A. Albrecht. Department of Infectious Diseases. New England Deaconess Hospital. I Autumn St., Boston. MA 02215. The Journal of Infectious Diseases
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© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0005$01.00
not ICP27, have been shown to trans-activate the HIV LTR [11], although the ability of these proteins to produce this effect depends on the cell line used [11-13]. Although transient expression assays using individual genes as inducer plasmids indicate that a given protein is able to affect ElV gene expression, the results of these tests may not accurately reflect the interactions of HIV with the full complement of HSV gene products as might occur in vivo. In an attempt to reproduce the in vivo situation more faithfully, we have superinfected cells persistently infected with HIV-I with intact wild-type HSV-1 and HSV-1 mutant viruses. ACH-2 cells were used as a model in vitro system to determine whether HSV can activate HIV gene expression in persistently HIV-infected cells. ACH-2 cells, subcloned from HIV-l-infected CEM cells, contain a single, integrated copy of proviral HIV-1 DNA [14]. In the resting state, these cells express low levels of viral RNA, primarily as singly (4.3 kb) and multiply spliced (2 kb) forms, with little or no full-length (9.2 kb) genomic mRNA [9, 15]. After stimulation with phorbol esters, which are known to induce activation of HIV-1 in these cells, not only is more total HIV RNA produced, but cells preferentially synthesize more of the 9.2-kb species [9, 15]. The altered pattern of RNA expression resulting from stimulation with activating agents mimics the transition from early to late transcription seen during productive infection [16]. The vast majority of ACH-2 cells are nonproductively infected, as evidenced by the cellular expression of HIV p24 antigen and by transmission electron microscopic studies [15]. Moreover, in the nonstimulated state, virion production can be detected in only 20% of these cells, whereas this level increases to nearly 100% within 21 h of induction [15]. Studies of peripheral blood mononuclear cells (PBMe)
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Heterologous viruses have been examined for their ability to accelerate the course of infection with the human immunodeficiency virus (HIV) type 1. In this study, ACH-2 cells persistently infected with HIV-1 exhibited augmented HIV-1 replication as a result of superinfection with herpes simplex virus (HSV) type 1. Using HSV-1 mutants with deletions in the genes encoding immediate-early proteins ICPO, ICP4, and ICP27, it was found that ICPO and ICP27, but not ICP4, were essential for up-regulation of HIV replication. Northern blot analysis showed that this activation of HIV was characterized by an initial rise in the level of the small, subgenomic (2.0 and 4.3 kb) mRNA species, followed by an increase in the level of unspliced genomic (9.2 kb) mRNA. Such a shift in transcriptional phase recapitulates the early-to-Iate transition seen in single-step growth curves of acute HIV -1 infection. Thus, HSV can activate HIV -1 from latency in ACH-2 cells, this activation of HIV is independent of productive HSV replication since the ~ICP4 deletion mutant is replication-incompetent, and this activation is evident as an increase in the steady-state levels of HIV transcripts.
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obtained from HIV-infected patients document the presence of HI V-specific RNA in all stages of HI V infection. By using the polymerase chain reaction, viral RNA could be detected in the PBMC of asymptomatic HIV -infected patients as well as those with frank AIDS [17]. Progressive HIV infection, determined by falling CD4 cell counts, was accompanied by the same preferential expression oflarge, unspliced genomic RNA seen in the in vitro models. These results challenge the prevailing notion of true virologic latency associated with HIV infection and imply that a cell line such as ACH-2 that is persistently infected with HIV may serve as a useful model to study mechanisms of up-regulation of HIV expression.
Materials and Methods
Beads were transferred to assay tubes, and 300 J,LL of o-phenylenediamine were added. The reaction was terminated after 30 min by addition of I N sulfuric acid. Color reaction was read with a spectrophotometer at 492 nm. HIV RNA isolation. Cellular RNA was prepared as previously described [16]. Briefly, RNA was harvested using guanadinium isothiocyanate denaturation followed by ultracentrifugation on a layer of cesium chloride. After resuspension of the pellet in RNase-free water, RNA was precipitated by treatment with 3 M NaOAc and 95%ethanol, then centrifuged at 12,000 g. The supernatant was discarded and the pellet resuspended in RNase-free water. RNA (20 J,Lg) was heat-denatured, electrophoresed on an agarose-formaldehyde gel, blotted onto nitrocellulose paper, washed, and baked at 80°C for 4 h. The DNA probe used for hybridization was the 511-bp fragment BglII of pWI3, which includes the polyadenylation sequence. HSV titer. Aliquots of cell culture underwent three freezethaw cycles to release intracellular virus, and serial dilutions of virus were plated in duplicate onto Vero cell monolayers. After a l-h adsorption at 37°C, the monolayers were overlaid with 0.5%agarose in Eagle MEM supplemented with 5% fetal bovine serum. Plates were incubated for 3-4 days, stained with neutral red, and reincubated for 24 h and plaques were counted.
Results KOS superinfection of ACH-2 cells. ACH-2 cells were fully permissive for productive infection with the KOS strain of HSV-I. Plotting HSV titer as a function of time after superinfection showed a rise in titer from 4.2 to 4.9 10glO pfu/ mL over 48 h (figure 1). Moreover, superinfection of ACH-2 cells with KOS resulted in increased HIV-1 expression, as determined by HIV -1 p24 supernatant antigen expression and RT activity (figures 2 and 3, respectively). By 72 h after superinfection with KOS, ACH-2 cells had p24 antigen levels of 22.8 ng/mL compared with 3.8 ng/ml, for mock-infected controls, almost a 10-fold rise in supernatant antigen concentration. Similar augmentation ofRT activity was seen by 72 h. ACH-2 cells superinfected with KOS had RT activity of 1.3 X 10 5 cpmjmL versus 6.0 X 10 3 cpmjmL for controls. AICP4 superinfection of ACH-2 cells. In an attempt to define the role of specific HSV transregulatory proteins in activation of HI V-I , we used KOS mutants with deletions in specific IE genes. The LlICP4 deletion mutant lacks both copies of the ICP4 gene, and since ICP4 is essential for virus growth, LlICP4 is replication incompetent [19]. When ACH2 cells were superinfected with the LlICP4 mutant, increases in supernatant p24 antigen expression and RT activity commensurate with those of wild-type virus were seen (figures 2 and 3). ACH-2 cells superinfected with the LlICP4 mutant had supernatant p24 antigen levels of 30.6 ngjmL at 72 h compared with 22.8 ngjmL for KOS-superinfected cells and 3.8 ng/mL for mock-infected controls. Levels ofRT activity rose to 8.0 X 104 cpmjmL at 72 h for cells superinfected with
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Cells. ACH-2 cells (CD4+ lymphoid cells), obtained from the AIDS Research and Reference Reagent Program (Rockville, MD), were maintained in RPMI 1640 medium supplemented with penicillin, streptomycin, t-glutamine. and 10% fetal bovine serum and were mycoplasma-free. Cell viability was determined daily by trypan blue dye exclusion. Viruses. Virus stocks included the wild-type strain ofHSV-1 (KOS), the ICPOdeletion mutant dlx3.1 (AICPO), the ICP4 deletion mutant dl20 (AICP4), and the ICP27 deletion mutant 5dll.2 (AICP27). KOS was used at an MOl of 1.0; all mutants were used at an MOl of 2.5. The deletion mutant of ICPO (dlx3.1) lacks the transcriptional start site and most of the ICPO coding sequence. It is replication competent and is phenotypically similar to wild-type virus when used at a high MOl [18]. The AICP4 (dI20) mutant lacks both copies of the ICP4 gene and as such is unable to synthesize early and late gene products [19-21]. This mutant is replication incompetent. The AICP27 (5dl1.2) deletion mutant lacks the transcriptional start site and portions of the ICP27 promoter and coding sequences [22] and is replication incompetent. Infection ofACH-2 cells. For each experiment, 2 X 107 cells were pelleted and exposed to I mL of virus stock or RPMI for 60 min at 37°C. The cells were then washed free of unadsorbed virus and resuspended in RPMI at a concentration of 5 X 105/mL. Reverse transcriptase (RT) activity. RT activity was determined by the method ofPoiesz et al. [23]. Virus was precipitated from culture supernatant with 0.4 M NaCl and 30% polyethylene glycol and placed on ice overnight. After centrifugation and resuspension, 0.9% Triton X-100 plus 1.5 MKCl was added to disrupt virions. An aliquot of this mixture was added to a solution containing 50 J,Lg of template primer poly(deoxyadenosine· deoxythymidine) or poly(adenosine· deoxythymidine)/ mL. [3H]dTTP (16 mCi/mol; NEN, Boston) was added, and counts were measured in an LS6800 liquid scintillation counter (Beckman Instruments, Brea, CA). HIV p24 antigen detection. HIV p24 antigen was measured using a sandwich solid-phase immunoassay (Abbott Laboratories, Abbott Park, IL). Culture supernatant (200 J,LL) was added to HIV antibody-coated polystyrene beads for 16-20 h. After a wash, the beads were incubated with rabbit anti-HIV antibody for 4 h, rewashed, and exposed to goat anti-rabbit IgG complexed to horseradish peroxidase for a final 2-h incubation.
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insufficient to induce HIV activation in these experiments. Viral superinfection did not produce visible cytopathic effect and viability remained at 90%, although the cultures were maintained for only 72 h after superinfection. HI V-specific RNA expression. It has previously been reported that activation of HIV in ACH-2 cells by such stimuli as phorbol esters and tumor necrosis factor (TNF) is accompanied by qualitative and quantitative changes in RNA expression. To determine whether these changes were also seen in HSV-infected ACH-2 cells, we examined the patterns of HIV RNA synthesis after superinfection ofACH-2 cells with KOS and all three mutants. HIV RNA levels began to in-
~ICP4 compared with 1.3 X 105 cpm/ml. for ACH-2 cells
superinfected with KOS and 6.0 X 103 cprn/ml. for controls. The ability of the ~ICP4 mutant to up-regulate HIV expression as efficiently as wild-type HSV indicates that neither the presence of ICP4 nor productive HSV infection per se was required for the activation of HIV-I in ACH-2 cells. ~ICPO and ~ICP27 superinfection of ACH-2 cells. Similar tests were conducted with the ~Icpb and ~ICP27 mutants. The ~ICPO mutant is replication competent because ICPO is not essential for virus growth [18]. This virus was unable to augment production ofeither HIV p24 antigen or RT after superinfection of ACH-2 cells (figures 2 and 3). The ~ICP27 mutant is replication incompetent because ICP27 is an essential replicative protein. This mutant expresses both ICPO and ICP4 [24] as well as the other IE proteins, ICP22 and ICP47. Mutant .1ICP27, like ~ICPO, had no effect on either RT or p24 antigen production (figures 2 and 3). Notably, these results also indicate that VPI6, a recognized trans-activator of IE HSV genes present in the virions of KOS and all three deletion mutants tested, was
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Time post-supertnfection (hours) Figure 3. Supernatant reverse transcriptase activity in cultures of ACH-2 cells mock-infected or infected with KOS strain ofHSYI or deletion mutant L1ICPO. L1ICP4. or L1ICP27.
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Figure 2. Supernatant HIY p24 antigen concentrations in cultures of ACH-2 cells mock-infected or infected with KOS strain of HSY-I or isogenic deletion mutant L1ICPO (dlx3.1), L1ICP4 (d 120), or L\ICP27 (5dlI.2).
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HIV Activated by HSV
crease 12 h after infection with KOS (figure 4) and continued to increase over the next 60 h. A differential accumulation of major RNA species occurred as a function of time after superinfection. The 2- and 4.3-kb RNAs appeared first, followed by the 9.2-kb species, which was ultimately expressed to a greater extent than the subgenomic RNA (figure 4). We next examined patterns of HIV RNA synthesis after superinfection of ACH-2 cells with dICPO, dICP4, and dICP27. Consistent with the HIV p24 antigen and RT data, HIV-specific RNA was detected in cells superinfected with dICP4 but not in cells infected with dICPO or dICP27 (figure 5). Activation oflatent HIV in ACH-2 cells was thus not detected after infection with HSV mutants containing deletions in the genes encoding ICPO or ICP27, whereas KOS and the dICP4 mutant substantially increased HIV replication and steady-state levels of HIV RNA.
At present, it is unclear precisely how HSV activates latent HIV in ACH-2 cells. Our results indicate that neither ICPO nor ICP27 alone is sufficient to activate HIV, since superinfection with dICP27 and ~ICPO failed to augment HIV replication. Furthermore, the ability of LlICP4, which possesses intact, functional ICP27 and ICPOgenes, to up-regulate HIV expression suggests that the combination ofICP27 and ICPO is likely necessary for this effect. To further address this possibility, transient transfection studies using HSV IE genes alone and in combination are planned. It is possible that ICPOand ICP27 interact directly with the proviral HIV LTR to enhance viral production. This is unlikely, however, as neither protein has been demonstrated to act through specific cis-acting elements in the promoters of genes known to be regulated by these two proteins [24, 37]. HSV may also activate HIV indirectly, through induction of cytokines or the transcription factor NF-KB. Since TNF affects baseline HIV expression in ACH-2 cells (via an autocrine mechanism) and mediates the increased HIV-l expression seen after treatment with phorbol esters [8], we have measured TNF levels after superinfection with HSV. At this point, our preliminary data show no difference in TNF levels after viral superinfection, but further studies to address this question are ongoing. It has been shown previously that HSV activation of an LTR CAT construct transfected into HeLa cells is associated with induction of NF-KB [38]. Because of the broad role of the transcription factor NF-KB in up-regulation of HIV expression, we have studied NF-KB induction after HSV superinfection. To date, we have failed to show concordance between the ability of KOS or any of the deletion mutants to induce the transcription factor NF-KB and up-regulation of HIV expression. We have previously demonstrated that during acute, productive HIV infection of the human T cell line CEM, the
Discussion The roles of ICP4, ICPO, and ICP27 in HSV replication have been studied extensively. ICP4, which is required for HSV replication, is a transcriptional activator of early and late genes of HSV, down-regulates IE gene expression (including its own), and can activate the HIV LTR [11,25-29]. ICPO, a protein not required for viral growth, is a potent trans-activator of all HSV genes tested to date, heterologous viral and cellular genes, and the HIV LTR [11, 18, 30]. The ICP27 gene encodes a multifunctional protein essential for HSV growth, with the capacity to either further activate or repress genes that are activated by ICPO, ICP4, or both [3135]. ICP27 may also modulate the expression of viral or cellular factors involved in host protein synthesis [36].
Figure 5. Expression of HIV-specific mRNA after infection of ACH-2 cells with strain KOS (K) or deletion mutant AICPO (0), AICP4 (4), or AICP27 (27). Mock-infected cells(M) showbaseline mRNA expression in unstimulated cells.
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Figure 4. Expression of HIV-specific mRNA after infection of ACH-2 cells with KOS strain ofHSV-1. Tubulin RNA hybridization (row T) shows uniform amounts of RNA in each lane. RNA made from H9 cellschronically infected with HIVstrain Ilia serves as positive control (C). Numbers at top indicate time (h) after superinfection; lane numbers are at bottom.
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stimulation of the production of human monokines capable ofregulating HIV-I expression. J ImmunoI1989;143:470-5. D'Addario M, Roulston A, Wainberg MA, Hiscott J. Coordinate enhancement ofcytokine gene expression in human immunodeficiency virus type I-infected promonocytic cells. J ViroI1990;64:6080-9. Folks TJ, Justement J, Kinter A, Oinarello CA, Fauci AS. Cytokine induced expression of HIV-I in a chronically infected promonocytic cell line. Science 1987;238:800-2. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor alpha functions in an autocrine manner in the induction ofhuman immunodeficiency virus expression. Proc Natl Acad Sci USA 1990;87:7825. Pomerantz RJ, Trono D, Feinberg MB, Baltimore O. Cells nonproductively infected with HIV-I exhibit an aberrant pattern of viral RNA expression: a molecular model for latency. Cell 1990;61: 1271-6. Rando RF, Pellet PE, Luciw PA, Bohan CA, Srinivasan A. Transactivation of human immunodeficiency virus by herpesviruses. Oncogene 1987;1:13-8. Ostrove JM, Leonard J, Weck KE, Rabson AB, Gendelman HE. Activation of the human immunodeficiency virus by herpes simplex type I. J ViroI1987;61:3726-32. Mosca JD, Bednarik DP, Raj NBK, et al. Activation ofhuman immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus I. Proc Nat! Acad Sci USA 1987;84:7408-12. Nabel GJ, Rice SA, Knipe DM, Baltimore D. Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells. Science 1988;239: 1299-302. Clouse KA, Powell D, Washington I, et al. Monokine regulation of human immunodeficiency virus-I expression in a chronically infected human T-cell clone. J Immunol 1989; 142:431-8. Michael NL, Morrow P, Mosca J, Vahey M, Burke DS, Redfield RR. Induction of human immunodeficiency virus type I expression in chronically infected cells is associated primarily with a shift in RNA splicing patterns. J ViroI1991;65:1291-303. Kim S, Byrn R, Groopman J, Baltimore D. Temporal aspects of ONA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression. J Virol 1989;63:3708-13. Michael NL, Vahey M, Burke OS, Redfield RR. Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type I infection in humans: evidence for viral replication in all stages of HIV disease. J Virol 1992;66:310-6. Sacks WR, Schaffer PA. Deletion mutants in the gene encoding the herpes simplex virus type I immediate-early protein ICPO exhibit impaired growth in cell culture. J ViroI1987;61:829-39. DeLuca NA, McCarthy AM, SchafferPA. Isolation and characterization of deletion mutants of herpes simplex virus type I in the gene encoding immediate-early regulatory protein, ICP4. J Virol 1985;56:558-70. Preston CM. Control of herpes simplex virus type I mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant ISK. J Virol 1979;29:275-84. Dixon RAF, Schaffer PA. Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type I immediate early protein VP175. J Virol 1980;36: 189-203. McCarthy AM, McMahon L, Schaffer PA. Herpes simplex virus type I ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J ViroI1989;63:18-27. Poiesz B, Ruscetti FW, Gagdar F, Bunn PA, Minna JO, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and
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parental cell line of ACH-2, HSV mutants lacking only the ICPO and ICP27 genes up-regulated HIV, while the LlICP4 mutant had no effect [39). In contrast, in the present study during activation oflatent HIV infection in ACH-2 cells, the LlICP4 mutant was a potent inducer of HIV expression, while neither the LlICPO nor LlICP27 mutant stimulated detectable HIV replication. A possible explanation for this discrepancy is that the ICP4 gene product enhances transcription only in the setting of productive infection, when HIV replication is not restricted. In latently infected cells, the quiescent provirus may depend on the presence of other viral or cellular proteins, whose activities or expression could be influenced by ICPO, ICP27, or both. What is clear at present is that regardless of the agent used to stimulate ACH-2 (mitogens, cytokines, or HSV infection), HIV activation in vivo and in vitro is marked by a substantial rise in the level of viral RNAs occurring in a predictable temporal sequence, with a shift toward preferential expression of the 9.2-kb mRNA species. Since rev expression may be responsible for the predominance of unspliced (9.2 kb) compared with small spliced RNA (2 and 4.3 kb) species, activation of the rev gene may be an important event in viral activation from latency in vitro. Unlike mitogens or cytokines, which broadly affect a number of cellular pathways, HSV gene products (such as ICPO) with their diverse transactivating functions likely affect regulation of proviral or host cell gene expression. Studies of virus-virus interaction with systems such as the one described here should promote a greater understanding of the pathogenesis of HIV at the cellular level. In a broader context, our results suggest that in people with clinically latent HIV infection, productive or even abortive infection with HSV, resulting either from acute de novo infection or reactivation from latency, may be involved in stimulating HIV replication, resulting in progression to AIDS. From a clinical standpoint, successful intervention in this and other virus-virus interactions may have a modulatory effect on the inexorable progression of HIV infection.
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32. Sekulovich RE. Leary K. Sandri-Goldin RM. The herpes simplex.virus type I alpha protein ICP27 can act as a trans-repressor or a tran~-acti vator in combination with ICP4 and ICPO.J ViroI1988;62:451 0-22. 33. Su L. Knipe OM. Herpes simplex virus alpha protein ICP27 can inhibit or augment viral gene transactivation. Virology 1989; 170:496-504. 34. Rice SA, Knipe OM. Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27. J Virol 1990;64: 1704-15. 35. Rice SA, Su L. Knipe OM. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J Virol 1989;63:3399-407. 36. McMahan L, Schaffer PA. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection. J ViroI1990;64:3471-85. 37. Gelman IH. Silverstein S. Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors. J Virol 1987;61 :2286-96. 38. Gimble JM. Duh E. Ostrove JM. Gendelman HE. Max EE. Rabson AB. Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type I is associated with induction ofa nuclear factor that binds to the NF-kB/core enhancer sequence. J Virol 1988;62:4104-12. 39. Albrecht MA, DeLuca NA, Byrn RA. Schaffer PA, Hammer SM. The herpes simplex virus immediate early protein. ICP4. is required to potentiate replication of human immunodeficiency virus in C04+ lymphocytes. J Virol 1989;63: 1861-8.
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29.
HIV Activated by HSV
Activation of Human Immunodeficiency Virus by Herpes Simplex Virus Marjorie P. Golden, Sunyoung Kim, Scott M. Hammer, Elizabeth A. Ladd, Priscilla A. Schaffer, Neal DeLuca, and Mary A. Albrecht
Division of Infectious Diseases, New England Deaconess Hospital, Laboratory ofTumor Virus Genetics, Dana Farber Cancer Institute, and Harvard Medical School, Boston, Massachusetts
Human immunodeficiency virus (HIV) type 1 is well established as the cause of AIDS [1-3]. In adults infected with HIV-1, clinical immunodeficiency appears after a mean incubation period of 8-10 years [4]. This prolonged clinical latency has led to speculation that, in certain individuals, infectious and other cofactors might affect disease progression. Up-regulation ofHIV expression in vitro is mediated by cytokines, protein kinase C activators, and heterologous viruses [5-10]. Antigens of several herpcsviruses, specifically cytomegalovirus (CMV) and Epstein-Barr virus, stimulate macrophages to produce cytokines, which in turn can up-regulate HIV expression in persistently infected monocytic and T lymphocytic cell lines [5]. The ability of heterologous DNA viruses to activate HIV has been studied most extensively using a chimeric plasmid containing the HIV long terminal repeat (LTR) linked to chloramphenicol acetyltransferase (CAT) as a reporter gene. When cells were transfected with this construct, CAT expression was increased after infection with herpes simplex virus (HSV) types 1 and 2, CMV, or varicella-zoster virus (VZV) [10]. In a transient cotransfection system, the HSV immediate-early (IE) regulatory proteins ICPOand ICP4, but
Received 30 January 1992; revised 13 April 1992. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, September 1991 (abstract 504). Financial support: National Institutes of Health (AI-OI0 15 to M.A., AI30897 to S.K., HL-4351 0 and -42112 to S.H .. AI-27431 to N.D., and CA20260 to P.S.); Department of Defense (DAMD 17-90-C-0106 to S.H.). Reprints or correspondence: Dr. Mary A. Albrecht. Department of Infectious Diseases. New England Deaconess Hospital. I Autumn St., Boston. MA 02215. The Journal of Infectious Diseases
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© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0005$01.00
not ICP27, have been shown to trans-activate the HIV LTR [11], although the ability of these proteins to produce this effect depends on the cell line used [11-13]. Although transient expression assays using individual genes as inducer plasmids indicate that a given protein is able to affect ElV gene expression, the results of these tests may not accurately reflect the interactions of HIV with the full complement of HSV gene products as might occur in vivo. In an attempt to reproduce the in vivo situation more faithfully, we have superinfected cells persistently infected with HIV-I with intact wild-type HSV-1 and HSV-1 mutant viruses. ACH-2 cells were used as a model in vitro system to determine whether HSV can activate HIV gene expression in persistently HIV-infected cells. ACH-2 cells, subcloned from HIV-l-infected CEM cells, contain a single, integrated copy of proviral HIV-1 DNA [14]. In the resting state, these cells express low levels of viral RNA, primarily as singly (4.3 kb) and multiply spliced (2 kb) forms, with little or no full-length (9.2 kb) genomic mRNA [9, 15]. After stimulation with phorbol esters, which are known to induce activation of HIV-1 in these cells, not only is more total HIV RNA produced, but cells preferentially synthesize more of the 9.2-kb species [9, 15]. The altered pattern of RNA expression resulting from stimulation with activating agents mimics the transition from early to late transcription seen during productive infection [16]. The vast majority of ACH-2 cells are nonproductively infected, as evidenced by the cellular expression of HIV p24 antigen and by transmission electron microscopic studies [15]. Moreover, in the nonstimulated state, virion production can be detected in only 20% of these cells, whereas this level increases to nearly 100% within 21 h of induction [15]. Studies of peripheral blood mononuclear cells (PBMe)
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Heterologous viruses have been examined for their ability to accelerate the course of infection with the human immunodeficiency virus (HIV) type 1. In this study, ACH-2 cells persistently infected with HIV-1 exhibited augmented HIV-1 replication as a result of superinfection with herpes simplex virus (HSV) type 1. Using HSV-1 mutants with deletions in the genes encoding immediate-early proteins ICPO, ICP4, and ICP27, it was found that ICPO and ICP27, but not ICP4, were essential for up-regulation of HIV replication. Northern blot analysis showed that this activation of HIV was characterized by an initial rise in the level of the small, subgenomic (2.0 and 4.3 kb) mRNA species, followed by an increase in the level of unspliced genomic (9.2 kb) mRNA. Such a shift in transcriptional phase recapitulates the early-to-Iate transition seen in single-step growth curves of acute HIV -1 infection. Thus, HSV can activate HIV -1 from latency in ACH-2 cells, this activation of HIV is independent of productive HSV replication since the ~ICP4 deletion mutant is replication-incompetent, and this activation is evident as an increase in the steady-state levels of HIV transcripts.
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obtained from HIV-infected patients document the presence of HI V-specific RNA in all stages of HI V infection. By using the polymerase chain reaction, viral RNA could be detected in the PBMC of asymptomatic HIV -infected patients as well as those with frank AIDS [17]. Progressive HIV infection, determined by falling CD4 cell counts, was accompanied by the same preferential expression oflarge, unspliced genomic RNA seen in the in vitro models. These results challenge the prevailing notion of true virologic latency associated with HIV infection and imply that a cell line such as ACH-2 that is persistently infected with HIV may serve as a useful model to study mechanisms of up-regulation of HIV expression.
Materials and Methods
Beads were transferred to assay tubes, and 300 J,LL of o-phenylenediamine were added. The reaction was terminated after 30 min by addition of I N sulfuric acid. Color reaction was read with a spectrophotometer at 492 nm. HIV RNA isolation. Cellular RNA was prepared as previously described [16]. Briefly, RNA was harvested using guanadinium isothiocyanate denaturation followed by ultracentrifugation on a layer of cesium chloride. After resuspension of the pellet in RNase-free water, RNA was precipitated by treatment with 3 M NaOAc and 95%ethanol, then centrifuged at 12,000 g. The supernatant was discarded and the pellet resuspended in RNase-free water. RNA (20 J,Lg) was heat-denatured, electrophoresed on an agarose-formaldehyde gel, blotted onto nitrocellulose paper, washed, and baked at 80°C for 4 h. The DNA probe used for hybridization was the 511-bp fragment BglII of pWI3, which includes the polyadenylation sequence. HSV titer. Aliquots of cell culture underwent three freezethaw cycles to release intracellular virus, and serial dilutions of virus were plated in duplicate onto Vero cell monolayers. After a l-h adsorption at 37°C, the monolayers were overlaid with 0.5%agarose in Eagle MEM supplemented with 5% fetal bovine serum. Plates were incubated for 3-4 days, stained with neutral red, and reincubated for 24 h and plaques were counted.
Results KOS superinfection of ACH-2 cells. ACH-2 cells were fully permissive for productive infection with the KOS strain of HSV-I. Plotting HSV titer as a function of time after superinfection showed a rise in titer from 4.2 to 4.9 10glO pfu/ mL over 48 h (figure 1). Moreover, superinfection of ACH-2 cells with KOS resulted in increased HIV-1 expression, as determined by HIV -1 p24 supernatant antigen expression and RT activity (figures 2 and 3, respectively). By 72 h after superinfection with KOS, ACH-2 cells had p24 antigen levels of 22.8 ng/mL compared with 3.8 ng/ml, for mock-infected controls, almost a 10-fold rise in supernatant antigen concentration. Similar augmentation ofRT activity was seen by 72 h. ACH-2 cells superinfected with KOS had RT activity of 1.3 X 10 5 cpmjmL versus 6.0 X 10 3 cpmjmL for controls. AICP4 superinfection of ACH-2 cells. In an attempt to define the role of specific HSV transregulatory proteins in activation of HI V-I , we used KOS mutants with deletions in specific IE genes. The LlICP4 deletion mutant lacks both copies of the ICP4 gene, and since ICP4 is essential for virus growth, LlICP4 is replication incompetent [19]. When ACH2 cells were superinfected with the LlICP4 mutant, increases in supernatant p24 antigen expression and RT activity commensurate with those of wild-type virus were seen (figures 2 and 3). ACH-2 cells superinfected with the LlICP4 mutant had supernatant p24 antigen levels of 30.6 ngjmL at 72 h compared with 22.8 ngjmL for KOS-superinfected cells and 3.8 ng/mL for mock-infected controls. Levels ofRT activity rose to 8.0 X 104 cpmjmL at 72 h for cells superinfected with
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Cells. ACH-2 cells (CD4+ lymphoid cells), obtained from the AIDS Research and Reference Reagent Program (Rockville, MD), were maintained in RPMI 1640 medium supplemented with penicillin, streptomycin, t-glutamine. and 10% fetal bovine serum and were mycoplasma-free. Cell viability was determined daily by trypan blue dye exclusion. Viruses. Virus stocks included the wild-type strain ofHSV-1 (KOS), the ICPOdeletion mutant dlx3.1 (AICPO), the ICP4 deletion mutant dl20 (AICP4), and the ICP27 deletion mutant 5dll.2 (AICP27). KOS was used at an MOl of 1.0; all mutants were used at an MOl of 2.5. The deletion mutant of ICPO (dlx3.1) lacks the transcriptional start site and most of the ICPO coding sequence. It is replication competent and is phenotypically similar to wild-type virus when used at a high MOl [18]. The AICP4 (dI20) mutant lacks both copies of the ICP4 gene and as such is unable to synthesize early and late gene products [19-21]. This mutant is replication incompetent. The AICP27 (5dl1.2) deletion mutant lacks the transcriptional start site and portions of the ICP27 promoter and coding sequences [22] and is replication incompetent. Infection ofACH-2 cells. For each experiment, 2 X 107 cells were pelleted and exposed to I mL of virus stock or RPMI for 60 min at 37°C. The cells were then washed free of unadsorbed virus and resuspended in RPMI at a concentration of 5 X 105/mL. Reverse transcriptase (RT) activity. RT activity was determined by the method ofPoiesz et al. [23]. Virus was precipitated from culture supernatant with 0.4 M NaCl and 30% polyethylene glycol and placed on ice overnight. After centrifugation and resuspension, 0.9% Triton X-100 plus 1.5 MKCl was added to disrupt virions. An aliquot of this mixture was added to a solution containing 50 J,Lg of template primer poly(deoxyadenosine· deoxythymidine) or poly(adenosine· deoxythymidine)/ mL. [3H]dTTP (16 mCi/mol; NEN, Boston) was added, and counts were measured in an LS6800 liquid scintillation counter (Beckman Instruments, Brea, CA). HIV p24 antigen detection. HIV p24 antigen was measured using a sandwich solid-phase immunoassay (Abbott Laboratories, Abbott Park, IL). Culture supernatant (200 J,LL) was added to HIV antibody-coated polystyrene beads for 16-20 h. After a wash, the beads were incubated with rabbit anti-HIV antibody for 4 h, rewashed, and exposed to goat anti-rabbit IgG complexed to horseradish peroxidase for a final 2-h incubation.
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insufficient to induce HIV activation in these experiments. Viral superinfection did not produce visible cytopathic effect and viability remained at 90%, although the cultures were maintained for only 72 h after superinfection. HI V-specific RNA expression. It has previously been reported that activation of HIV in ACH-2 cells by such stimuli as phorbol esters and tumor necrosis factor (TNF) is accompanied by qualitative and quantitative changes in RNA expression. To determine whether these changes were also seen in HSV-infected ACH-2 cells, we examined the patterns of HIV RNA synthesis after superinfection ofACH-2 cells with KOS and all three mutants. HIV RNA levels began to in-
~ICP4 compared with 1.3 X 105 cpm/ml. for ACH-2 cells
superinfected with KOS and 6.0 X 103 cprn/ml. for controls. The ability of the ~ICP4 mutant to up-regulate HIV expression as efficiently as wild-type HSV indicates that neither the presence of ICP4 nor productive HSV infection per se was required for the activation of HIV-I in ACH-2 cells. ~ICPO and ~ICP27 superinfection of ACH-2 cells. Similar tests were conducted with the ~Icpb and ~ICP27 mutants. The ~ICPO mutant is replication competent because ICPO is not essential for virus growth [18]. This virus was unable to augment production ofeither HIV p24 antigen or RT after superinfection of ACH-2 cells (figures 2 and 3). The ~ICP27 mutant is replication incompetent because ICP27 is an essential replicative protein. This mutant expresses both ICPO and ICP4 [24] as well as the other IE proteins, ICP22 and ICP47. Mutant .1ICP27, like ~ICPO, had no effect on either RT or p24 antigen production (figures 2 and 3). Notably, these results also indicate that VPI6, a recognized trans-activator of IE HSV genes present in the virions of KOS and all three deletion mutants tested, was
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Time post-supertnfection (hours) Figure 3. Supernatant reverse transcriptase activity in cultures of ACH-2 cells mock-infected or infected with KOS strain ofHSYI or deletion mutant L1ICPO. L1ICP4. or L1ICP27.
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Figure 2. Supernatant HIY p24 antigen concentrations in cultures of ACH-2 cells mock-infected or infected with KOS strain of HSY-I or isogenic deletion mutant L1ICPO (dlx3.1), L1ICP4 (d 120), or L\ICP27 (5dlI.2).
JID 1992;166 (September)
HIV Activated by HSV
crease 12 h after infection with KOS (figure 4) and continued to increase over the next 60 h. A differential accumulation of major RNA species occurred as a function of time after superinfection. The 2- and 4.3-kb RNAs appeared first, followed by the 9.2-kb species, which was ultimately expressed to a greater extent than the subgenomic RNA (figure 4). We next examined patterns of HIV RNA synthesis after superinfection of ACH-2 cells with dICPO, dICP4, and dICP27. Consistent with the HIV p24 antigen and RT data, HIV-specific RNA was detected in cells superinfected with dICP4 but not in cells infected with dICPO or dICP27 (figure 5). Activation oflatent HIV in ACH-2 cells was thus not detected after infection with HSV mutants containing deletions in the genes encoding ICPO or ICP27, whereas KOS and the dICP4 mutant substantially increased HIV replication and steady-state levels of HIV RNA.
At present, it is unclear precisely how HSV activates latent HIV in ACH-2 cells. Our results indicate that neither ICPO nor ICP27 alone is sufficient to activate HIV, since superinfection with dICP27 and ~ICPO failed to augment HIV replication. Furthermore, the ability of LlICP4, which possesses intact, functional ICP27 and ICPOgenes, to up-regulate HIV expression suggests that the combination ofICP27 and ICPO is likely necessary for this effect. To further address this possibility, transient transfection studies using HSV IE genes alone and in combination are planned. It is possible that ICPOand ICP27 interact directly with the proviral HIV LTR to enhance viral production. This is unlikely, however, as neither protein has been demonstrated to act through specific cis-acting elements in the promoters of genes known to be regulated by these two proteins [24, 37]. HSV may also activate HIV indirectly, through induction of cytokines or the transcription factor NF-KB. Since TNF affects baseline HIV expression in ACH-2 cells (via an autocrine mechanism) and mediates the increased HIV-l expression seen after treatment with phorbol esters [8], we have measured TNF levels after superinfection with HSV. At this point, our preliminary data show no difference in TNF levels after viral superinfection, but further studies to address this question are ongoing. It has been shown previously that HSV activation of an LTR CAT construct transfected into HeLa cells is associated with induction of NF-KB [38]. Because of the broad role of the transcription factor NF-KB in up-regulation of HIV expression, we have studied NF-KB induction after HSV superinfection. To date, we have failed to show concordance between the ability of KOS or any of the deletion mutants to induce the transcription factor NF-KB and up-regulation of HIV expression. We have previously demonstrated that during acute, productive HIV infection of the human T cell line CEM, the
Discussion The roles of ICP4, ICPO, and ICP27 in HSV replication have been studied extensively. ICP4, which is required for HSV replication, is a transcriptional activator of early and late genes of HSV, down-regulates IE gene expression (including its own), and can activate the HIV LTR [11,25-29]. ICPO, a protein not required for viral growth, is a potent trans-activator of all HSV genes tested to date, heterologous viral and cellular genes, and the HIV LTR [11, 18, 30]. The ICP27 gene encodes a multifunctional protein essential for HSV growth, with the capacity to either further activate or repress genes that are activated by ICPO, ICP4, or both [3135]. ICP27 may also modulate the expression of viral or cellular factors involved in host protein synthesis [36].
Figure 5. Expression of HIV-specific mRNA after infection of ACH-2 cells with strain KOS (K) or deletion mutant AICPO (0), AICP4 (4), or AICP27 (27). Mock-infected cells(M) showbaseline mRNA expression in unstimulated cells.
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Figure 4. Expression of HIV-specific mRNA after infection of ACH-2 cells with KOS strain ofHSV-1. Tubulin RNA hybridization (row T) shows uniform amounts of RNA in each lane. RNA made from H9 cellschronically infected with HIVstrain Ilia serves as positive control (C). Numbers at top indicate time (h) after superinfection; lane numbers are at bottom.
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References I. Barre-Sinoussi F, Chermann Jc, Rey F, et al. Isolation of a T-lyrnphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1989;220:868-71. 2. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 1984;224:500-3. 3. Levy JA, Hoffman AD, Kramer SM, Lanois JA, Shimabukuro JM, Oskiro LS. Isolation oflymphocytopathic retroviruses from San Francisco patients with AIDS. Science 1984;225:840-2. 4. Lifson AR, Rutherford GW, Jaffe HW. The natural history of HIV infection. J Infect Dis 1988;158:1360-7. 5. Clouse KA, Robbins PB, Fernie B, Ostrove JM, Fauci AS. Viral antigen
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stimulation of the production of human monokines capable ofregulating HIV-I expression. J ImmunoI1989;143:470-5. D'Addario M, Roulston A, Wainberg MA, Hiscott J. Coordinate enhancement ofcytokine gene expression in human immunodeficiency virus type I-infected promonocytic cells. J ViroI1990;64:6080-9. Folks TJ, Justement J, Kinter A, Oinarello CA, Fauci AS. Cytokine induced expression of HIV-I in a chronically infected promonocytic cell line. Science 1987;238:800-2. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor alpha functions in an autocrine manner in the induction ofhuman immunodeficiency virus expression. Proc Natl Acad Sci USA 1990;87:7825. Pomerantz RJ, Trono D, Feinberg MB, Baltimore O. Cells nonproductively infected with HIV-I exhibit an aberrant pattern of viral RNA expression: a molecular model for latency. Cell 1990;61: 1271-6. Rando RF, Pellet PE, Luciw PA, Bohan CA, Srinivasan A. Transactivation of human immunodeficiency virus by herpesviruses. Oncogene 1987;1:13-8. Ostrove JM, Leonard J, Weck KE, Rabson AB, Gendelman HE. Activation of the human immunodeficiency virus by herpes simplex type I. J ViroI1987;61:3726-32. Mosca JD, Bednarik DP, Raj NBK, et al. Activation ofhuman immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus I. Proc Nat! Acad Sci USA 1987;84:7408-12. Nabel GJ, Rice SA, Knipe DM, Baltimore D. Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells. Science 1988;239: 1299-302. Clouse KA, Powell D, Washington I, et al. Monokine regulation of human immunodeficiency virus-I expression in a chronically infected human T-cell clone. J Immunol 1989; 142:431-8. Michael NL, Morrow P, Mosca J, Vahey M, Burke DS, Redfield RR. Induction of human immunodeficiency virus type I expression in chronically infected cells is associated primarily with a shift in RNA splicing patterns. J ViroI1991;65:1291-303. Kim S, Byrn R, Groopman J, Baltimore D. Temporal aspects of ONA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression. J Virol 1989;63:3708-13. Michael NL, Vahey M, Burke OS, Redfield RR. Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type I infection in humans: evidence for viral replication in all stages of HIV disease. J Virol 1992;66:310-6. Sacks WR, Schaffer PA. Deletion mutants in the gene encoding the herpes simplex virus type I immediate-early protein ICPO exhibit impaired growth in cell culture. J ViroI1987;61:829-39. DeLuca NA, McCarthy AM, SchafferPA. Isolation and characterization of deletion mutants of herpes simplex virus type I in the gene encoding immediate-early regulatory protein, ICP4. J Virol 1985;56:558-70. Preston CM. Control of herpes simplex virus type I mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant ISK. J Virol 1979;29:275-84. Dixon RAF, Schaffer PA. Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type I immediate early protein VP175. J Virol 1980;36: 189-203. McCarthy AM, McMahon L, Schaffer PA. Herpes simplex virus type I ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J ViroI1989;63:18-27. Poiesz B, Ruscetti FW, Gagdar F, Bunn PA, Minna JO, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and
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parental cell line of ACH-2, HSV mutants lacking only the ICPO and ICP27 genes up-regulated HIV, while the LlICP4 mutant had no effect [39). In contrast, in the present study during activation oflatent HIV infection in ACH-2 cells, the LlICP4 mutant was a potent inducer of HIV expression, while neither the LlICPO nor LlICP27 mutant stimulated detectable HIV replication. A possible explanation for this discrepancy is that the ICP4 gene product enhances transcription only in the setting of productive infection, when HIV replication is not restricted. In latently infected cells, the quiescent provirus may depend on the presence of other viral or cellular proteins, whose activities or expression could be influenced by ICPO, ICP27, or both. What is clear at present is that regardless of the agent used to stimulate ACH-2 (mitogens, cytokines, or HSV infection), HIV activation in vivo and in vitro is marked by a substantial rise in the level of viral RNAs occurring in a predictable temporal sequence, with a shift toward preferential expression of the 9.2-kb mRNA species. Since rev expression may be responsible for the predominance of unspliced (9.2 kb) compared with small spliced RNA (2 and 4.3 kb) species, activation of the rev gene may be an important event in viral activation from latency in vitro. Unlike mitogens or cytokines, which broadly affect a number of cellular pathways, HSV gene products (such as ICPO) with their diverse transactivating functions likely affect regulation of proviral or host cell gene expression. Studies of virus-virus interaction with systems such as the one described here should promote a greater understanding of the pathogenesis of HIV at the cellular level. In a broader context, our results suggest that in people with clinically latent HIV infection, productive or even abortive infection with HSV, resulting either from acute de novo infection or reactivation from latency, may be involved in stimulating HIV replication, resulting in progression to AIDS. From a clinical standpoint, successful intervention in this and other virus-virus interactions may have a modulatory effect on the inexorable progression of HIV infection.
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cultured lymphocytes ofa patient with cutaneous T-cell lymphoma. Proc Nat! Acad Sci USA 1980;77:7415-9. Rice SA. Knipe OM. Gene-specific transactivation by herpes simplex virus type I alpha protein ICP27. J ViroI1988;62:3814-23. DeLuca NA, Schaffer PA, Physical and functional domains of the herpes simplex transcriptional regulatory protein ICP4. J Virol 1988;62:732-43. Everett RD. Trans-activation of transcription by herpes virus products: requirement of two HSV-I immediate-early polypeptides for maximum activity. EMBO J 1984;3:3135-41. Gelman IH. Silverstein S. Identification of immediate-early genes from herpes simplex that transactivate the virus thymidine kinase gene. Proc Nat! Acad Sci USA 1985;82:5265-9. Mavromara-Navos P. Silver S, Hubenthal-VossJ. McKnight MC, Roizman B. Regulation of herpes simplex virus type I genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late promoters. Virology 1986; 149:152-64. O'Hare P. Hayward GS. Three trans-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation. J Virol 1985;56:723-33. Everett RD. The products of herpes simplex virus type I (HSV-I) immediate-early genes I. 2. and 3 can activate HSV-I gene expression in trans. J Gen Virol 1986;67:2507-13. Sacks WR. Greene CC, Aschman OP. Schaffer PA, Herpes simplex virus type I ICP27 is an essential regulatory protein. J Virol 1985;55:796-805.
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32. Sekulovich RE. Leary K. Sandri-Goldin RM. The herpes simplex.virus type I alpha protein ICP27 can act as a trans-repressor or a tran~-acti vator in combination with ICP4 and ICPO.J ViroI1988;62:451 0-22. 33. Su L. Knipe OM. Herpes simplex virus alpha protein ICP27 can inhibit or augment viral gene transactivation. Virology 1989; 170:496-504. 34. Rice SA, Knipe OM. Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27. J Virol 1990;64: 1704-15. 35. Rice SA, Su L. Knipe OM. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J Virol 1989;63:3399-407. 36. McMahan L, Schaffer PA. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection. J ViroI1990;64:3471-85. 37. Gelman IH. Silverstein S. Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors. J Virol 1987;61 :2286-96. 38. Gimble JM. Duh E. Ostrove JM. Gendelman HE. Max EE. Rabson AB. Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type I is associated with induction ofa nuclear factor that binds to the NF-kB/core enhancer sequence. J Virol 1988;62:4104-12. 39. Albrecht MA, DeLuca NA, Byrn RA. Schaffer PA, Hammer SM. The herpes simplex virus immediate early protein. ICP4. is required to potentiate replication of human immunodeficiency virus in C04+ lymphocytes. J Virol 1989;63: 1861-8.
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29.
HIV Activated by HSV
