Onset Of Viral Gene Expression Research Articles

Single-cell herpes simplex virus type 1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics.

Single-cell analyses of viral infections reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of herpes simplex virus type 1 (HSV-1) infection of neurons at the single-cell level. We used micrometer-scale Matrigel beads, termed microgels, to culture individual murine superior cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are encapsulated in individual media-in-oil droplets with a dual-fluorescent reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. These techniques for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ134.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ134.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ134.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ134.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Promoting Simultaneous Onset of Viral Gene Expression Among Cells Infected with Herpes Simplex Virus-1.

Synchronous viral infection facilitates the study of viral gene expression, viral host interactions, and viral replication processes. However, the protocols for achieving synchronous infections were hardly ever tested in proper temporal resolution at the single-cell level. We set up a fluorescence-based, time lapse microscopy assay to study sources of variability in the timing of gene expression during herpes simplex virus-1 (HSV-1) infection. We found that with the common protocol, the onset of gene expression within different cells can vary by more than 3 h. We showed that simultaneous viral genome entry to the nucleus can be achieved with a derivative of the previously characterized temperature sensitive mutant tsB7, however, this did not improve gene expression synchrony. We found that elevating the temperature in which the infection is done and increasing the multiplicity of infection (MOI) significantly promoted simultaneous onset of viral gene expression among infected cells. Further, elevated temperature result in a decrease in the coefficient of variation (a standardized measure of dispersion) of viral replication compartments (RCs) sizes among cells as well as a slight increment of viral late gene expression synchrony. We conclude that simultaneous viral gene expression can be improved by simple modifications to the infection process and may reduce the effect of single-cell variability on population-based assays.

The Heme Metabolite Carbon Monoxide Facilitates KSHV Infection by Inhibiting TLR4 Signaling in Endothelial Cells.

Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and certain rare B cell lymphoproliferative disorders. KSHV infection of endothelial cells (EC) in vitro increases expression of the inducible host-encoded enzyme heme oxygenase-1 (HO-1), which is also strongly expressed in KS tumors. HO-1 catalyzes the rate-limiting step in the conversion of heme into iron, biliverdin and the gasotransmitter carbon monoxide (CO), all of which share anti-apoptotic, anti-inflammatory, pro-survival, and tumorigenic activities. Our previous work has shown that HO-1 expression in KSHV-infected EC is characterized by a rapid yet transient induction at early times post-infection, followed by a sustained upregulation co-incident with establishment of viral latency. These two phases of expression are independently regulated, suggesting distinct roles for HO-1 in the virus life cycle. Here, we investigated the role of HO-1 during acute infection, prior to the onset of viral gene expression. The early infection phase involves a series of events that culminate in delivery of the viral genome to the nucleus. Primary infection also leads to activation of host innate immune effectors, including the pattern recognition receptor TLR4, to induce an antiviral response. It has been shown that TLR4-deficient EC are more susceptible to KSHV infection than wild-type controls, suggesting an important inhibitory role for TLR4 in the KSHV life cycle. TLR4 signaling is in turn subject to regulation by several virus-encoded immune evasion factors. In this report we identify HO-1 as a host protein co-opted by KSHV as part of a rapid immune evasion strategy. Specifically, we show that early HO-1 induction by KSHV results in increased levels of endogenous CO, which functions as a TLR4 signaling inhibitor. In addition, we show that CO-mediated inhibition of TLR4 signaling leads to reduced expression of TLR4-induced antiviral genes, thus dampening the host antiviral response and facilitating KSHV infection. Conversely, inhibition of HO-1 activity decreases intracellular CO, enhances the host antiviral response and inhibits KSHV infection. In conclusion, this study identifies HO-1 as a novel innate immune evasion factor in the context of KSHV infection and supports HO-1 inhibition as a viable therapeutic strategy for KS.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

P53 elevation in human cells halt SV40 infection by inhibiting T-ag expression.

SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression.

U L 31 of Herpes Simplex Virus 1 Is Necessary for Optimal NF-κB Activation and Expression of Viral Gene Products

Previous results suggested that the U(L)31 gene of herpes simplex virus 1 (HSV-1) is required for envelopment of nucleocapsids at the inner nuclear membrane and optimal viral DNA synthesis and DNA packaging. In the current study, viral gene expression and NF-κB and c-Jun N-terminal kinase (JNK) activation of a herpes simplex virus mutant lacking the U(L)31 gene, designated ΔU(L)31, and its genetic repair construct, designated ΔU(L)31-R, were studied in various cell lines. In Hep2 and Vero cells infected with ΔU(L)31, expression of the immediate-early protein ICP4, early protein ICP8, and late protein glycoprotein C (gC) were delayed significantly. In Hep2 cells, expression of these proteins failed to reach levels seen in cells infected with ΔU(L)31-R or wild-type HSV-1(F) even after 18 h. The defect in protein accumulation correlated with poor or no activation of NF-κB and JNK upon infection with ΔU(L)31 compared to wild-type virus infection. The protein expression defects of the U(L)31 deletion mutant were not explainable by a failure to enter nonpermissive cells and were not complemented in an ICP27-expressing cell line. These data suggest that pU(L)31 facilitates initiation of infection and/or accelerates the onset of viral gene expression in a manner that correlates with NF-κB activation and is independent of the transactivator ICP27. The effects on very early events in expression are surprising in light of the fact that U(L)31 is designated a late gene and pU(L)31 is not a virion component. We show herein that while most pUL31 is expressed late in infection, low levels of pU(L)31 are detectable as early as 2 h postinfection, consistent with an early role in HSV-1 infection.

Adenovirus-induced alterations in host cell gene expression prior to the onset of viral gene expression

In this report, we have studied gene expression profiles in human primary lung fibroblasts (IMR-90) during the very early phase of an adenovirus infection. Eight out of twelve genes with known functions encoded transcription factors linked to two major cellular processes; inhibition of cell growth (ATF3, ATF4, KLF4, KLF6 and ELK3) and immune response (NR4A1 and CEBPB), indicating that the earliest consequences of an adenovirus infection are growth arrest and induction of an immune response. A time course analysis showed that the induction of these immediate-early response genes was transient and suppressed after the onset of the adenovirus early gene expression.

Posttranscriptional Suppression of Interleukin-6 Production by Human Cytomegalovirus

Human cytomegalovirus (HCMV) has evolved multiple strategies for suppression of the antiviral response of the infected cell. DNA array technology has revealed that HCMV clearly regulates host gene expression during the course of a productive infection by enhancing, sustaining, or suppressing steady-state levels of cellular transcripts. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in the immune response to infection. Here we report a detailed study of the effects of HCMV infection on IL-6 expression by human fibroblasts. UV-inactivated virus was found to induce high levels of IL-6 mRNA and protein expression, and IL-6 mRNA remained abundant in cells 16 h after inoculation even though the level of ongoing IL-6 transcription was not significantly enhanced. In lytic HCMV infections, the onset of viral gene expression resulted in two apparently antagonistic effects on IL-6 expression: (i) transcriptional activation, mediated at least in part by the IE2p86 protein, and (ii) posttranscriptional suppression mediated by destabilization of IL-6 mRNA. Transcriptional activation was outweighed by the suppressive effect, such that cells undergoing productive infection produced less IL-6 than cells challenged with inactivated virus. Suppression of IL-6 expression was independent of the viral IL-10 homologue, cmvIL-10. Destabilization of IL-6 mRNA was observed to coincide with the enhanced expression and aberrant intracellular localization of HuR, an mRNA-binding protein known to interact with IL-6 and other mRNAs containing 3' AU-rich elements. Our data suggest a novel mechanism for gene regulation by HCMV at the posttranscriptional level.

Autographa californica M nucleopolyhedrovirus early GP64 synthesis mitigates developmental resistance in orally infected noctuid hosts.

The unusual early synthesis of the Autographa californica M nucleopolyhedrovirus (AcMNPV) budded virus (BV) structural protein GP64 is an important virulence factor during oral infection of Heliothis virescens larvae. Considering the breadth of the AcMNPV host range, the importance of early GP64 synthesis in orally infected permissive hosts (Trichoplusia ni and Spodoptera exigua) from subfamilies other than that of H. virescens was assessed. An AcMNPV recombinant, having wild-type early and late GP64 synthesis, was compared with one in which only late GP64 synthesis occurred. Early GP64 synthesis was found to have more of an effect on virulence in orally inoculated T. ni than S. exigua and that virulence was dependent on two factors: the ability of the host to slough occlusion-derived virus (ODV)-infected midgut cells and the rapidity with which BV was transmitted to the tracheal cells. In both host species, insects inoculated orally with the control virus transmitted BV to tracheal cells hours before those inoculated with the gp64 temporal mutant. Moreover, with early GP64 synthesis, the lag between the onset of viral gene expression in midgut and tracheal cells was only 3-4 h, supporting the conclusion that in these insects, the first systemic infections arose from ODV-derived nucleocapsids repackaged as BV. These results provide further empirical proof that early GP64 synthesis is a component of a unique and selectively advantageous baculovirus infection strategy for exploiting larval lepidopterans by counteracting developmental resistance.

Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes.

Primary cytomegalovirus (CMV) infection and reactivation of persistent CMV are associated with significant morbidity and mortality in immunocompromised individuals. Although recovery from CMV disease is correlated with the development of CMV-specific cytotoxic T lymphocytes (CTL), the major viral target antigens to which the response is directed are ill-defined, though they may comprise viral structural elements. We now identify the CMV matrix protein pp65 as a significant target antigen for CD8+ class I major histocompatibility complex (MHC)-restricted CMV-specific CTL derived from the peripheral blood of four of five latently infected individuals. CMV-specific CTL recognition of pp65 on target cells occurs prior to the onset of viral gene expression and persists throughout the duration of the replicative cycle. Recognition in the absence of viral gene expression suggests that abundant viral protein enters the normal trafficking pathway upon viral penetration and is readily made available to MHC molecules for presentation at the cell surface. Thus pp65 specific CTL may represent an important effector population for early control and limitation of CMV infection and disease. The observation that CMV-specific CTL can be induced in vitro using peptide fragments derived from pp65 supports the future use and manipulation of this and similar effector populations in a clinical setting.