Research ArticleAIDS/HIVVirology Open Access | 10.1172/JCI164317

Carl A. Pierce,1 Lip Nam Loh,1 Holly R. Steach,2 Natalia Cheshenko,1 Paula Preston-Hurlburt,3 Fengrui Zhang,4 Stephanie Stransky,5 Leah Kravets,1 Simone Sidoli,5 William Philbrick,4 Michel Nassar,6 Smita Krishnaswamy,2,7 Kevan C. Herold,3,4 and Betsy C. Herold1,8

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Pierce, C. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Loh, L. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Steach, H. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Cheshenko, N. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Preston-Hurlburt, P. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Zhang, F. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Stransky, S. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Kravets, L. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Sidoli, S. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Philbrick, W. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Nassar, M. in: JCI | PubMed | Google Scholar

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Krishnaswamy, S. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Herold, K. in: JCI | PubMed | Google Scholar |

1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.

2Department of Computational Biology,

3Department of Immunobiology, and

4Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

5Department of Biochemistry and

6Department of Otorhinolaryngology–Head and Neck Surgery, Albert Einstein College of Medicine, New York, New York, USA.

7Department of Genetics, Yale School of Medicine, New Haven, Connecticut, USA.

8Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

Address correspondence to: Betsy C. Herold, Departments of Pediatrics and Microbiology-Immunology, Albert Einstein College of Medicine 1225 Morris Park Avenue, Bronx, New York 10461, USA. Phone: 718.839.7460; Email: betsy.herold@einsteinmed.edu. Or to: Kevan C. Herold, Departments of Immunobiology and Medicine, Yale School of Medicine 300 George Street, New Haven, Connecticut 06520, USA. Phone: 203.785.6507; Email: kevan.herold@yale.edu. LNL’s present address is: Lytica Therapeutics, Cambridge, Massachusetts, USA.

Authorship note: KCH and BCH contributed equally to this work.

Find articles by Herold, B. in: JCI | PubMed | Google Scholar

Authorship note: KCH and BCH contributed equally to this work.

Published April 20, 2023 - More info

Published in Volume 133, Issue 11 on June 1, 2023
J Clin Invest. 2023;133(11):e164317. https://doi.org/10.1172/JCI164317.
© 2023 Pierce et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published April 20, 2023 - Version history
Received: August 8, 2022; Accepted: April 17, 2023 View PDF Abstract

Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2–infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2–infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.

Introduction

Herpes simplex virus type 2 (HSV-2) is a common coinfection in persons living with human immunodeficiency virus type 1 (HIV-1) and is considered one of the most important cofactors driving the global HIV-1 epidemic. Epidemiologic studies consistently demonstrate that prevalent and incident HSV-2 infections are associated with an increased risk of HIV-1 acquisition and transmission (1). Moreover, among HIV-1/HSV-2–coinfected individuals, prevalent HSV-2 is associated with higher HIV-1 genital and plasma viral loads, which increase following HSV-2 outbreaks (2, 3). Subclinical shedding of HSV-2 is also associated with expanded HIV-1 tissue reservoirs and an increased divergence from the most recent common ancestor (4).

Most studies of the HIV-1 and HSV-2 syndemic have focused on local responses to HSV-2 that promote HIV-1 acquisition. For example, in HIV-1–seronegative (HIV–) individuals, symptomatic HSV-2 reactivation was associated with an influx of immune cells including activated CD4+ T cells at the site of lesions that may persist for a prolonged time (5). Even in the absence of clinical reactivation, an increase in activated CD4+, CCR5+ T cells in foreskin tissue and female genital tract samples and increased expression of T cell activation markers in the peripheral blood have been observed in HSV-2–seropositive (HSV-2+) compared with HSV-2–seronegative (HSV-2–) individuals (6, 7). These activated T cells could serve as targets for new HIV-1 infection but do not explain why coinfection is associated with an increased frequency of HIV-1 viremic episodes even among patients on antiretroviral therapy (8–10).

While asymptomatic HSV-2 infection was associated with the recruitment and persistence of activated CD4+ T cells in cervicovaginal samples obtained from HIV– women, it was not associated with a significant difference in proinflammatory cytokines or chemokines in cervicovaginal fluid (6). Consistent with these observations, we also identified few significant differences in cytokine, chemokine, or antimicrobial peptide concentrations in genital tract secretions obtained from HIV-1+ women who were or were not coinfected with HSV-2 (11). However, we did find significant phenotypic differences in peripheral blood CD4+ (but not CD8+) T cells comparing coinfected versus HIV-1+, HSV-2– women (12). Specifically, we found increased frequency of CCR5+, CXCR4+, PD-1+, and CD69+ and decreased frequency of CCR10+ and CCR6+ CD4+ T cells. These changes were associated with higher levels of cell-associated HIV-1 DNA. Paradoxically, IL-32, a proinflammatory cytokine, was lower in subpopulations of CD4+ T cells in HSV-2+ versus HSV-2– women, and the addition of recombinant IL-32γ blocked HIV reactivation in CD4+ T cells treated with phytohemagglutinin (PHA) (12, 13). Other studies found that siRNA targeting IL-32 resulted in an increase in HIV replication (13). Together these findings suggested that the phenotypic changes in CD4+ T cells, including the decrease in IL-32γ associated with HSV-2, may promote HIV-1 reactivation and/or replication. However, the molecular mechanisms underlying these changes and their effects on HIV-1 reactivation are not known.

Activated CD4+ T cells are susceptible to HSV-2 infection in vitro (14), and virus has been detected in CD4+ T cells isolated from vesicle fluid of genital lesions and within biopsies of HSV-2 skin lesions (15). The recruitment and persistence of activated CD4+ T cells to the genital mucosa during HSV-2 reactivation (5) and the potential for activated peripheral blood CD4+ T cells to be exposed to HSV during episodes of transient HSV-2 viremia (16–18) prompted us to postulate that HSV-2 might have direct or indirect bystander effects on CD4+ T cells to promote HIV-1 reactivation and/or replication. We therefore analyzed the effects of HSV-2 infection of activated primary CD4+ T cells and an immortalized human CD4+ T cell line model of HIV-1 latency (Jurkat 2D10 cells) (19). We identified changes in the HSV-2–infected and bystander CD4+ T cells that were associated with HIV-1 reactivation, including upregulation of the long noncoding MALAT1. The ability of HSV-2 to trigger HIV latency reversal was reduced in MALAT1-knockout 2D10 cells.

Results

HSV-2 productively infects activated CD4+ T cells. To confirm the previously reported susceptibility of activated CD4+ T cells and Jurkat T cells to HSV-2 infection, we infected Jurkat or primary human CD4+ T cells with HSV-2(333ZAG), which expresses GFP under a CMV promoter (20). We observed a dose-dependent increase in the percentage of GFP+ Jurkat cells following exposure to a multiplicity of infection (MOI) of 1, 5, or 10 PFU/cell of 21.85%, 40.75%, and 45.90%, respectively (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI164317DS1). Exposure of stimulated (anti-CD3/anti-CD28) primary human CD4+ T cells (isolated from n = 20 different donor leukopaks) to HSV-2(333ZAG) (MOI = 1) resulted in 27.69% ± 11.79% (mean ± SD) GFP+ cells, whereas unstimulated cells were resistant (0.28% ± 0.16%) (Figure 1A and Supplemental Figure 1B). Productive HSV-2 infection of activated CD4+ T cells was confirmed using a low-passage clinical isolate, HSV-2(SD90), at MOI = 0.001 and quantification of infectious virus released into culture supernatants at 6, 24, and 48 hours post-infection (hpi) by plaque assay. Kinetics of infection in activated CD4+ T and HaCaT cells (human keratinocytes) were similar, although viral yields were lower (log10 PFU/mL = 6.40 vs. 4.12 at 48 hpi, P < 0.001) (Figure 1B). The mean CD4+ T cell viability was 86.92% (range 78.00%–93.00%) 6 hours, 95.25% (92.50%–97.00%) 24 hours, and 91.76% (84.50%–98.58%) 48 hours after infection (Figure 1C). Consistent with our previous observations, 24 hpi with 3 different isolates of HSV-2 (SD90, 333ZAG, or G) (MOI = 1), there was a 0.39 ± 0.24 log10-fold decrease in IL32 and a 0.53 ± 0.19 log10-fold increase in CD69 expression relative to mock-infected cells (P < 0.001) (Figure 1D) (12).

Figure 1

HSV-2 productively infects activated primary CD4+ T cells and downregulates IL-32 and upregulates CD69 expression. (A) Primary human CD4+ T cells isolated from healthy donor leukopaks were stimulated by CD3/CD28 cross-linking for 72 hours (n = 22) or left unstimulated (n = 4) and then incubated with GFP-expressing HSV-2(333ZAG) at an MOI of 1 PFU/cell for 2 hours, washed, and cultured for a further 22 hours, and the percentage of GFP+ cells was quantified by flow cytometry. (B) Primary anti-CD3/CD28–stimulated CD4+ T cells (filled symbols) or HaCaT cells (open symbols) were infected with HSV-2(SD90) (MOI = 0.001 PFU/cell), and at the indicated times, the amount of infectious virus released into the culture supernatants was quantified by plaque assays conducted in duplicate on Vero cells. Values of 0 PFU were set to zero before log transformation. Viral yields from the 2 different cell types were compared at 24 and 48 hours. ***P < 0.001, Mann-Whitney test. (C) CD4+ T cell viability following HSV-2 infection as in B was determined by vital dye exclusion (n = 6 donors). (D) Anti-CD3/CD28–stimulated CD4+ T cells from n = 3 different donors were infected with the indicated isolates of HSV-2 at an MOI of 1 PFU/cell, and at 24 hpi, IL32 and CD69 gene expression was quantified by RT-qPCR. Results are presented as log10 fold change (FC) relative to mock-infected CD4+ T cells.

We further characterized CD4+ T cells that were susceptible to HSV-2 infection by flow cytometry. Peripheral blood CD4+ T cells (stimulated with anti-CD3/CD28) were mock-infected or infected with HSV-2(SD90) (MOI = 1). Infected cells were identified by staining with a fluorophore-conjugated antibody against HSV-2 glycoprotein B (anti-gB). The gB+ cells were more likely to be CD45RO+ (58.13% ± 5.37% CD45RO+ vs. 21.90% ± 5.64% CD45RO–, mean ± SD, P < 0.0001) (Figure 2A) and to express Tbet (64.65% vs. 34.26%, P < 0.01), RORγT (61.47% vs. 13.97%, P < 0.01), and Bcl6 (69.09% vs. 20.91%, P < 0.0001) (Figure 2B). The frequency of GATA3+ cells was not different in the gB+ versus the gB– cells (5.75% vs. 4.49%), and the frequency of FoxP3+ cells was lower (2.97% vs. 16.76%, P < 0.05) (Figure 2C).

Figure 2

HSV-2–infected cells are preferentially CD45RO+ CD4+ T cells and express the transcription factors T-bet, RORγT, and Bcl6. (A) CD4+ T cells from n = 5 healthy donor leukopaks were stimulated for 72 hours by CD3/CD28 cross-linking, infected with HSV-2(SD90) (MOI = 1 PFU/cell), and cultured for a further 24 hours, and then stained for glycoprotein B (gB) and for CD45RO. The percentage of gB+ cells in the total CD4+ T cell (CD45RO–/+) population, CD4+CD45RO– population, and CD4+CD45RO+ population was quantified by flow cytometry. *P < 0.05, ***P < 0.001, ****P < 0.0001, 1-way ANOVA. (B) Representative flow cytometry plots of HSV-2 gB (y axis) and transcription factor (x axis) staining with electronic gates placed on CD4+ T cells. (C) Cells were infected as in A and stained for gB and for the indicated transcription factors (TFs). The percentages of gB+ (infected) and gB– (bystander) cells expressing the indicated markers were compared by paired t test; *P < 0.05, **P < 0.01, ****P < 0.0001.

HSV-2 infection of CD4+ T cells promotes HIV reactivation and replication. To determine whether HSV-2 impacted HIV replication, CD4+ T cells isolated from HIV+ donors were activated with PHA and then infected with HSV-2(SD90) (MOI = 1) for 48 hours (n = 3). We used PHA rather than anti-CD3/CD28 for these studies because identifying HIV-infected cells harboring replication-competent virus in the peripheral blood is challenging and PHA has been shown to more consistently promote HIV replication (12). The cells were stained with anti-gB and anti-p24 antibodies to identify HSV-2– and HIV-1–infected cells, respectively, and analyzed by flow cytometry (Figure 3A). With the HSV-2 infection after PHA stimulation, there was an increase in the mean fluorescence intensity (MFI) of p24 staining in 3 of 3 donors compared with activation with PHA alone (P = 0.056, paired t test) (Figure 3B). In all 3 donors, the percentage of p24+ cells was greater in the gB+ than in the gB– cells.

Figure 3

HSV-2 infection of T cells promotes HIV reactivation and replication. (A and B) CD4+ T cells from HIV+ donors with plasma viral loads (PVLs) of 3,210 (donor 1, orange), 23,860 (donor 2, blue), and 44,700 (donor 3, green) copies/mL, respectively, were stimulated with PHA for 24 hours and then mock-infected or infected with HSV-2(SD90) (MOI = 1 PFU/cell) and, 48 hpi, were stained for HSV-2 gB and HIV-1 p24 (A). The mean fluorescence intensity (MFI) of p24 was determined; the bar represents the median (P = 0.056, paired t test, PHA + HSV-2 vs. PHA alone) (B). (C and D) 2D10 cells were exposed to live or UV-inactivated (UV) HSV-2(G) (C) or to strain G, 4674, SD90, or heat-inactivated (HI) G (D) (MOI = 1 PFU/cell). HIV ltr gene expression was measured by RT-qPCR relative to mock-infected samples 24 hpi (n = 3–6 each, ****P < 0.0001 comparing live vs. UV in C [t test] and each strain versus HI virus by 1-way ANOVA in D). (E) 2D10 cells were infected with HSV-2(G) at an MOI of 1 or 10 PFU/cell, and at 24 hpi, cells were fixed and stained with anti-gB antibody (red). Nuclei were stained with DAPI (blue), and HIV-reactivating cells were detected by eGFP (green). Representative images (original magnification, 63×1.4) from 2 independent experiments are shown. (F) 2D10 cells were infected with HSV-2(G) (MOI = 1 PFU/cell) for 8 hours and washed, and then fresh medium or medium supplemented with PHA (10 μg/mL) or TNF (10 ng/mL) was added. The cells were fixed and stained 24 hours after HSV-2 exposure. Representative images (original magnification, 63×1.4) from 2 independent experiments are shown. (G) The numbers of gB+ (red), eGFP+ (green), and gB+/eGFP+ (merge) cells and total cells (blue) were quantified in 5 randomly selected fields in E and F using Cell Counter ImageJ software (NIH); pie charts show relative proportions.

These findings suggest that HSV-2 promoted HIV-1 replication, but the frequency of HIV-1–infected primary CD4+ cells was (as expected) low and PHA activation was needed. To address these experimental limitations, we used the Jurkat-derived 2D10 cell line model of HIV latency (19). 2D10 cells were exposed, without prior stimulation, to live or UV-inactivated HSV-2(G) (MOI = 1) and HIV ltr expression measured 24 hpi. There was a 1 log10-fold increase in ltr expression in response to live, but not UV-inactivated, HSV-2(G) in comparison with uninfected cells (Figure 3C). To exclude the possibility that this response was isolate specific, we exposed 2D10 cells to 2 additional HSV-2 clinical isolates (4674 and SD90). All 3 isolates resulted in an at least 1 log10-fold increase in ltr expression in comparison with uninfected cells (Figure 3D). To validate the findings and to compare the response in HSV-2–infected and bystander cells, we conducted additional confocal microscopy studies. 2D10 cells were infected with HSV-2(G) (MOI = 1 or 10 PFU/cell) and, 24 hpi, fixed and stained for gB (conjugated to Alexa Fluor 647, red). HIV-reactivating cells were identified by expression of enhanced GFP (eGFP) (19). At MOI = 1, 13.3% of cells expressed gB and eGFP (reactivating, HSV-2–infected cells), 17.2% were GFP+ only (reactivating bystanders), and 7.1% were gB+ only (n = 406 cells, 5 random fields). At MOI = 10, 43.7% stained for both gB and GFP, 5.9% for GFP only, and 22.7% for gB only (n = 238 cells, 5 fields) (Figure 3, E and G).

To test whether HSV-2 exposure may interfere with the response to other reactivating agents, we first infected the cells with HSV-2 (MOI = 1) for 8 hours and then, after washing, treated the cells with medium alone or medium containing PHA or TNF, and analyzed the cells by confocal microscopy 24 hpi. HSV-2 itself triggered HIV reactivation in 15.24% ± 0.1% of the cells, and the response increased significantly (P < 0.05, 1-way ANOVA) to 41.75% ± 4.85% and 47.43% ± 5.42% when PHA or TNF, respectively, was subsequently added (mean ± SEM, n = 2 independent experiments) (Figure 3, F and G). The percentage of reactivating bystanders (eGFP+, gB–) increased from 10.9% ± 0.15% to 21.15% ± 0.95% (P < 0.01) and to 28.85% ± 2.45% (P < 0.05) when PHA or TNF was added to the cultures, respectively. The percentage of HIV-1+HSV-2+ dually stained cells also increased from 4.5% ± 0.25% to 20.08% ± 4.45% and 11.7% ± 7.7%, respectively. Together these results suggest that HSV-2 triggers HIV reactivation in HSV-2–infected and bystander CD4+ T cells in a dose-dependent manner and may act in concert with other activating stimuli.

HSV-2 upregulates genes associated with HIV latency reversal and downregulates restriction factors. To identify molecular mechanisms that may account for how HSV-2 promotes HIV-1 latency reversal and/or HIV-1 replication, we conducted RNA-Seq studies of HSV-2–infected CD4+ T cells that were isolated from leukopaks of 5 HIV-uninfected donors. The cells were activated with anti-CD3/CD28 for 72 hours and then mock-infected or infected with HSV-2(333ZAG) (MOI = 1). After 24 hours, the cells were separated into GFP+ and GFP– populations by fluorescence-activated cell sorting (FACS). A mean of 33.82% (SD = 8.60) of cells were GFP+.

We identified 7,841 genes whose expression was increased significantly (log2 fold change > 1 and adjusted P < 0.01) and 6,013 whose expression was decreased significantly (log2 fold change > 1 and adjusted P < 0.01) comparing GFP+ and mock-infected cells. We also found significant changes in the GFP– (bystander) cells with an increase in expression of 1,217 genes and a decrease in expression of 994 genes compared with mock-infected cells, although the magnitude of changes was smaller. Gene set enrichment analysis of Gene Ontology (GO) terms identified 16 ontologies with adjusted P values less than 0.05 that were relevant to viral processes (Supplemental Table 1). We focused on the 3 largest (GO:0009615, response to virus; GO:0019080, viral gene expression; and GO:0019058, viral life cycle), which together comprised 632 unique genes and had considerable overlap. We performed principal component analysis of these genes. The first principal component (PC) captured 78.5% of variance and differentiated the GFP+ samples from the GFP– or mock-infected samples; PC2 captured 11.7% of variance and largely contributed to the separation of GFP– and mock-infected samples (Figure 4A). The genes contributing most strongly to PC1 and PC2 are shown in Supplemental Figure 2 and Supplemental Table 2.

Figure 4

Transcriptional changes in HSV-2–infected CD4+ T cells. CD4+ T cells isolated from leukopaks of 5 healthy donors were stimulated for 72 hours by CD3/CD28 cross-linking, infected with HSV-2(ZAG) at an MOI of 10 in biological duplicate, and sorted on GFP expression, and RNA was isolated for RNA-Seq. (A) Principal component analysis using the genes included in GO:0009615, 0019080, and 0019058. (B) Volcano plots comparing expression of a subset of genes identified in our bulk RNA-Seq data selected based on their known association with HIV reactivation and replication, comparing GFP+ versus mock (left) and GFP– versus mock (right). Select genes of interest are demarcated in red. Dotted vertical lines indicate fold change greater than 2. All demarcated genes were significant (adjusted P < 0.05). A3G, APOBEC3G. (C) Normalized count data for selected genes related to HIV infection, latency reversal, or the interferon response. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Wald test via DESeq2.

We compared expression of a subset of these genes, focusing on those that have been previously associated with HIV-1 latency reversal, replication, and/or pathogenesis (Figure 4, B and C, and Supplemental Table 3) (21). Among the differentially expressed genes, we found significantly increased expression in the GFP+ compared with mock-inf