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The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Adult↗

Chemokine receptor trafficking and viral replication.

Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, but chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways. Ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Part of the mechanism through which chemokines protect cells from HIV infection is through ligand-induced internalization of the specific chemokine receptor co-receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane-trafficking pathways and the influence of these interactions on viral replication.

Amino Acid Sequence↗

Interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.

The human cytomegalovirus UL82-encoded pp71 protein is required for efficient virus replication and immediate-early gene expression when cells are infected at a low multiplicity. Functions attributed to pp71 include the ability to enhance the infectivity of viral DNA, bind to and target hypophosphorylated Rb family member proteins for degradation, drive quiescent cells into the cell cycle, and bind to the cellular protein hDaxx. Using UL82 mutant viruses, we demonstrate that the LXCXD motif within pp71 is not necessary for efficient virus replication in fibroblasts, suggesting that pp71's ability to degrade hypophosphorylated Rb family members and induce quiescent cells into the cell cycle is not responsible for the growth defect associated with a UL82 deletion mutant. However, UL82 mutants that cannot bind to hDaxx are unable to induce immediate-early gene expression and are severely attenuated for viral replication. These results indicate that the interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.

Adaptor Proteins, Signal Transducing↗

Rhesus macaque resistance to mucosal simian immunodeficiency virus infection is associated with a postentry block in viral replication.

Elucidation of the host factors which influence susceptibility to human immunodeficiency virus or simian immunodeficiency virus (SIV) infection and disease progression has important theoretical and practical implications. Rhesus macaque 359, a vaccine control animal, resisted two successive intravaginal challenges with SIV(mac251) and failed to seroconvert. Here, after an additional intrarectal SIVmac32H challenge, macaque 359 remained highly resistant to infection. Viral RNA (10(6) copies/ml) was observed in plasma only at week 2 postchallenge. Virus isolation and proviral DNA were positive only once at week eight postchallenge. The animal remained seronegative and cleared SIV in vivo. Its blood and lymph node cells obtained at 49 weeks after intrarectal challenge did not transmit SIV to a naive macaque. We found that the resistance of macaque 359 to SIV infection was not due to a high level of CD8(+) suppressor activity but to an inherent resistance of its CD4(+) T cells. To elucidate the basis for the unusually strong resistance of macaque 359 to SIV infection in vivo and in vitro, we investigated early events of viral infection and replication in CD4(+) cells of macaque 359, including expression and mutation screening of SIV coreceptors and analysis of viral entry and reverse transcription. Mutation screening revealed no genetic alteration in SIV coreceptors. PCR analysis revealed a significant delay in production of early in vitro reverse transcription intermediates in macaque 359 cells compared to susceptible controls, but cell fusion assays showed that SIV entered the CD4(+) CCR5(+) cells of macaque 359 as readily as cells of macaques susceptible to SIV infection. Our results suggest that the resistance of macaque 359 to SIV infection is due to a postentry block in viral replication and implicate a cellular inhibitory mechanism in its CD4(+) T cells. Identification of this host mechanism will help further elucidate the biochemistry of reverse transcription and may suggest therapeutic strategies. Determining the prevalence of this host resistance mechanism among macaques may lead to better design of SIV pathogenesis and vaccine studies.

Animals↗

YB-1 relocates to the nucleus in adenovirus-infected cells and facilitates viral replication by inducing E2 gene expression through the E2 late promoter.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.

Active Transport, Cell Nucleus↗

Efficacy of a replication-selective adenovirus against ovarian carcinomatosis is dependent on tumor burden, viral replication and p53 status.

Intraperitoneal (i.p.) recurrence of cisplatin-refractory and p53 mutant ovarian cancer is a major clinical problem, despite surgery and chemotherapy. dl1520 (ONYX-015) is an E1B-55 kDa gene-deleted adenovirus engineered selectively to replicate in and destroy cancer cells lacking functional p53. However, a correlation between efficacy and p53 function has not been definitively studied in vivo to date, and efficacy following i.p. administration had not been previously described. We therefore carried out experiments to address these issues in three nude mouse-human ovarian carcinomatosis xenograft models. Intraperitoneal treatment with dl1520 led to complete tumor eradication and/or significantly improved survival in two p53(-) nude mouse-human ovarian tumor xenograft models. OVCAR3 i.p. xenografts underwent complete regressions in 11 of 12 mice (versus one of seven controls; P = 0.001), while mice bearing cisplatin-resistant A2780 tumors had significantly improved survival versus controls (P = 0.05). In contrast, the A2780 p53(+) ovarian cancer xenograft was resistant to dl1520. The efficacy of i.p. dl1520 in the p53(-) models correlated strongly with tumor burden present at the time of treatment initiation, and no efficacy was seen with non-replicating/UV-inactivated dl1520. Selectively replicating viruses such as dl1520 hold promise as i.p. therapies for p53-deficient and chemotherapy-resistant ovarian carcinomas. A phase I clinical trial of i.p. dl1520 (ONYX-015) is underway in patients with cisplatin-resistant ovarian carcinoma.

Adenoviridae↗

Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication.

We previously described a persistent infection established by the lymphotropic minute virus of mice in mouse L cells at the level of the cell population (D. Ron, P. Tattersall, and J. Tal, J. Virol. 52:63-69, 1984). This carrier state is maintained by a series of consecutive phenotypic changes which take place in both the cells and the virus and is cured spontaneously after 150 to 200 cell generations (D. Ron and J. Tal, J. Virol. 55:424-430, 1985). We show here that the cure was caused by the selection of virus-resistant cells in the culture. The resistance of these survivor cells to virus replication was due to an intracellular block. Infection of a spontaneously cured culture with the fibrotropic parental minute virus of mice resulted in a restrictive infection in which the viral replicative-form DNA was formed and amplified, but the synthesis of single-stranded progeny DNA was markedly reduced. The lymphotropic strain was blocked in these cells at an earlier stage, with little or no amplification of viral replicative-form DNA observed. These data indicate that the replication of minute virus of mice requires host-coded helper functions in at least two stages of its growth cycle.

Animals↗

In vitro transcription of bacteriophage phi 29 DNA: inhibition of early promoters by the viral replication protein p6.

The effect of the phi 29 protein p6 on the in vitro initiation of transcription from the main phi 29 promoters was studied. Protein p6 interfered with the transcription from the early promoters C1 and C2, located at the right end of the phi 29 genome. Transcription initiated at the early promoters A1, A2b, and A2c and at the late promoter A3, located at the left region of the viral genome, was not affected by protein p6. These results suggest that protein p6 might play a dual role, acting as a negative modulator of some phi 29 early promoters apart from being a positive modulator of the viral DNA replication.

Bacteriophages↗

The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies.

Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and S2. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the S2 gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the S2 gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344-8348, 1998). We now describe in vivo studies that examine the evolution and role of the S2 gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that the S2 gene is highly conserved during persistent infection and that deletion of the S2 gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional S2 gene. These data indicate that the EIAV S2 gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of S2 influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAV S2 that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.

Amino Acid Sequence↗

Respiratory syncytial virus-induced chemokine production: linking viral replication to chemokine production in vitro and in vivo.

Respiratory syncytial virus (RSV) is a negative-sense, single-strand RNA virus that can initiate severe bronchiolitis in infants, as well as in elderly adults. Although RSV preferentially infects and replicates in the airway epithelium, studies have shown that RSV has the ability to infect and, to a limited extent, replicate in alveolar macrophages. In the present study, we sought to characterize the RSV-induced chemokine production in vitro and in vivo, because chemokines have been shown to contribute to both the inflammation and pathophysiology of disease. Our results show that RSV-infected airway epithelial cells and alveolar macrophages display differential profiles of chemokine production: airway epithelial cells produce CCL2/monocyte chemoattractant protein-1, CCL5/RANTES, CXCL10/gamma interferon inducible protein-10, and kerotinocyte cytokine (KC); and alveolar macrophages up-regulate CCL5 and macrophage inflammatory protein (MIP)-2 after RSV infection. In vivo, we observed the induction of CCL2, CCL3/MIP-1 alpha, CCL5, CXCL10, and KC after RSV infection. In the present study, we also addressed the necessity for viral infection and/or replication in chemokine induction by use of ultraviolet (UV)-inactivated RSV, as well as RSV inhibitors of binding/infection and replication, that is, NMSO3, a sulfated sialyl lipid compound, and ribavirin, respectively. Our results suggest that viral replication is necessary for optimal chemokine production.

Animals↗

Role of hepatitis B, C, and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal core promoter mutations on viral replicative interference.

The interactions among hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) were studied by measuring HBV-DNA and HCV-RNA levels and by determining the influence of viral genotypes and mutations in HBV basal core promoter (BCP) and precore regions. We included 65 consecutive patients, 25 HBV/HCV, 18 HBV/HDV, and 22 HBV/HCV/HDV. Controls consisted of 55 patients with chronic HBV and 55 with chronic HCV infection. HBV-DNA and HCV-RNA levels were lower in coinfections than in single infections (P <.05). HBV/HCV coinfection was associated with lower HBV viremia (8.2 x 10(4) copies/mL) and lower HCV-RNA levels (7 x 10(5) IU/mL), than the corresponding control group (P <.05), with more marked decrease in HBV replication (P <.05). Moreover, in HBV/HCV coinfection and in triple coinfection we observed an inverse relationship between HBV-DNA and HCV-RNA levels (P <.05). HBV/HDV coinfection was associated with lower HBV viremia (2.5 x 10(4) copies/mL) than that found in HBV infection (P <.05). Patients with triple coinfection showed lower HBV-DNA and HCV-RNA levels than control groups (P <.05). Prevalence of precore mutations was lower in HCV coinfections (P <.05). No significant association was observed between HCV-RNA levels and HBV precore mutations, BCP mutations or HBV genotypes, or between HBV-DNA levels and HCV genotypes (P <.05). In conclusion, HCV exhibited stronger inhibitory action in the reciprocal inhibition seen in HBV/HCV coinfection. HDV was the dominant virus in HBV/HDV coinfection and in triple coinfection, and had a greater unfavorable influence on HCV than on HBV replication. The reciprocal inhibition of viral replication seemed to be little influenced by the inherent genomic factors studied.

Adult↗

HIV-1-specific CD4+ T cell responses in chronically HIV-1 infected blippers on antiretroviral therapy in relation to viral replication following treatment interruption.

The impact of transient viral load blips on anti-HIV-1 immune responses and on HIV-1 rebound following treatment interruption (TI) is not known. Clinical and immunological parameters were measured during 40 weeks of antiretroviral therapy (ART) and following TI in an observational cohort of 16 chronically HIV-1-infected subjects with or without observed viral load blips during ART. During therapy, blips in seven subjects were associated with higher anti-HIV-1 (p24) CD4+ T cell lymphoproliferative responses (p = 0.04), without a significant difference in T cell activation or total anti-HIV-1 CD8+ T cell interferon-gamma (IFN-gamma) responses when compared to nine matched non-blippers. Therapy interruption resulted in a significantly higher viral rebound in blippers by 8 week despite retention of higher lymphoproliferative p24 responses (p = 0.01) and a rise in CD3+ T cell activation (p = 0.04) and anti-HIV-1 CD8+ T cell responses in blippers by week 4 when compared to non-blippers. Past week 4 of interruption, therapy re-initiation criteria were also met by a higher frequency in blippers by week 14 (p < 0.04) with no difference between groups by week 24. These data support that blippers have higher anti-HIV lymphoproliferative responses while on ART but experience equal to higher viral rebound as compared to matched non-blippers upon TI.

Anti-HIV Agents↗

Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture.

The sequence encoding the viral leader proteinase Npro was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187. The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus. At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA. This RNA lacked the same viral genes as the subgenomic RNA which has been found in all cytopathogenic CSFV strains analyzed so far, but it maintained the ubiquitin sequence.

Animals↗

Site of suppression of Banzi viral replication by an antiviral factor released from Aedes albopictus cells persistently infected with Banzi virus.

The ability of the antiviral factor present in culture medium of Aedes albopictus cells persistently infected with the flavivirus, Banzi virus, to inhibit the replication of Banzi virus was examined. The anti-Banzi viral factor did not inhibit the uncoating of the virion. Levels of viral RNA were markedly reduced in mosquito cells treated with the antiviral factor. Syntheses of negative-strand and of positive-strand viral RNA species were inhibited to approximately the same extent. This inhibition was virus-specific in that the anti-Banzi viral factor had no effect on the synthesis of viral RNA in mosquito cells infected with either Japanese encephalitis or Eastern equine encephalitis viruses. The anti-Banzi viral factor inhibited the in vitro Banzi viral RNA synthesis but not that of Eastern equine encephalitis virus or of Japanese encephalitis virus.

Aedes↗

Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt.

DNA replication of Epstein-Barr virus (EBV) during the productive phase of the life cycle of this herpesvirus depends on the cis-acting element oriLyt. It consists of two essential domains, the upstream and the downstream component. Whereas the upstream component contains several DNA-binding motifs for the viral activator protein BZLF1, the downstream component is known to be the binding site of several cellular proteins. We identified cellular transcription factors that bind synergistically to a functionally relevant subsequence of the downstream component, the TD element. Two of these transcription factors, ZBP-89 and Sp1, stimulate replication as shown by protein fusions with the GAL4 DNA-binding domain and a single GAL4 DNA-binding motif inserted into the TD element. In protein binding assays, we observed an interaction of Sp1 and ZBP-89 with the viral DNA polymerase and its processivity factor. Our data indicate that cellular transcriptional activators tether viral replication proteins to the lytic origin via direct protein-protein interactions to assemble the viral replication complex at oriLyt.

Base Sequence↗

Minus sense transcripts of brome mosaic virus RNA-3 intercistronic region interfere with viral replication.

Interference with virus replication through the use of defective viral sequences is providing new insight to replication strategies and novel approaches for induced resistance. Because replication of brome mosaic virus (BMV) is potentiated by the intercistronic region of RNA-3, we examined the effect of adding various (-)sense RNAs corresponding to this region in co-transfections with wild type BMV RNAs. Progeny accumulation in barley protoplasts transfected with RNAs 1+2 was decreased by 90% in the presence of (-)RNA-3 delta HindIII, the longest (-)sense transcript tested, and by 85% when RNA-3 was also present. This trans interference was concentration dependent, and the use of deletion derivatives of (-)RNA-3 delta HindIII revealed that previously identified regulatory sequences within the intercistronic region were responsible for the observed interference. These deletion mutants were found to be of differing stabilities and several served as effective substrates for host-encoded polymerase to yield complementary (+)strands. Indeed, it is possible that the copying of viral RNA by the host polymerase serves as a hybrid arrest mechanism for discriminating against viral RNA functions. However, neither the ability of these sequences to serve as templates for host polymerase nor their (+)strand products contributed to the interference phenomenon, which may provide a new approach for engineering resistance to viral infection.

Capsid↗

Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection.

Animals↗