Single-Cell Study Reveals Three Distinct Fates of Macrophages Infected by Gammaherpesvirus

Gammaherpesviruses are masters of persistence. The human members of this family, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, establish lifelong infections and are linked to chronic inflammation and cancer, especially in people with weakened immune systems. Yet one of the most basic questions about how these viruses interact with the immune cells they first encounter has remained surprisingly murky. A new study published in iScience by Gabrielle Vragel, Eric Clambey, Linda van Dyk, and colleagues at the University of Colorado now provides the most detailed picture to date of what happens when a gammaherpesvirus infects macrophages, the innate immune cells that serve as both sentinels against pathogens and, it turns out, willing hosts for the virus.

Because the human gammaherpesviruses infect only their natural hosts with fidelity, researchers rely on murine gammaherpesvirus 68, or MHV68, a genetically and phenotypically faithful mouse model that recapitulates the key features of its human relatives. Previous work had established that MHV68 infects macrophages, but the literature contained contradictory findings: some studies reported active lytic replication, in which the virus hijacks the cell to produce new infectious particles, while others documented quiescent latent infection, and still others suggested that infection in these cells was abortive or unstable over time. The new study set out to resolve this ambiguity by examining infection at single-cell resolution, both in living animals and in a controlled cell culture system.

The team began by infecting immunocompetent C57BL/6J mice with a reporter virus carrying a beta-lactamase enzyme fused to LANA, the latency-associated nuclear antigen encoded by the ORF73 gene. This fusion protein allows infected cells to be identified and purified with high sensitivity using flow cytometry, because the enzymatic activity of beta-lactamase cleaves a fluorescent substrate inside the cell. Sixteen hours after infection, the researchers sort-purified infected peritoneal macrophages and subjected them to single-cell RNA sequencing, a technique that captures the complete transcriptional profile of individual cells, including any viral transcripts they harbor.

The results revealed a striking and unexpected transcriptional landscape. Among more than 9,100 infected macrophages analyzed, the vast majority carried viral RNA reads that made up less than 0.1 percent of their total transcripts, indicating a highly restricted infection. The dominant viral signal came from the ORF75 locus, a set of three duplicated genes, ORF75A, ORF75B, and ORF75C, that is conserved across the gammaherpesvirus family. More than half of the infected macrophages showed detectable transcription from at least one of these ORF75 genes, while most other viral genes were detected only sporadically. This pattern is remarkable because ORF75 transcripts have previously been observed in Kaposi’s sarcoma lesions and in Epstein-Barr virus-transformed B cells, but selective ORF75 transcription had not been reported during MHV68 infection of macrophages. The researchers confirmed that the reads mapped specifically to each ORF75 gene rather than representing unspliced ORF73 RNA, since the three ORF75 genes have diverged substantially at their 3-prime ends.

In contrast to this dominant restricted population, a tiny subset of macrophages, just 25 cells out of more than 9,500, displayed a completely different signature: abundant expression of canonical lytic cycle genes including ORF7, ORF54, ORF57, ORF58, and ORF61, detected in every one of these cells. These rare cells were clearly initiating productive viral replication in vivo, while their neighbors remained transcriptionally silent apart from ORF75. The finding demonstrates that even within a single cell type, at a single early time point, gammaherpesvirus infection produces fundamentally different outcomes in different cells.

To dissect these outcomes under controlled conditions, the researchers turned to the J774 macrophage cell line, comparing infection with the permissive 3T12 fibroblast line. In fibroblasts, MHV68 underwent robust lytic replication, destroying the monolayer within 72 hours. In J774 cells, the virus readily entered and initiated expression of the ORF73 immediate-early gene, with infection frequencies comparable to fibroblasts, but viral genome replication and production of infectious particles barely exceeded input levels. Quantitative RT-PCR showed that ORF73 transcripts were reduced roughly tenfold in macrophages relative to fibroblasts, while the early gene ORF6 and the late gene M7 were decreased more than a hundredfold, revealing progressively greater deficits at later stages of the lytic cycle.

Flow cytometry-based reporter assays confirmed this picture. A virus expressing GFP under a lytic promoter marked about 8 percent of infected fibroblasts but fewer than 1 percent of macrophages. Markers of lytic protein expression, including the viral regulator of complement activation encoded by ORF4 and the phosphorylated histone H2AX, which is targeted by the viral protein kinase ORF36 during replication, appeared in roughly 45 percent of infected fibroblasts but only about 2 percent of infected macrophages at 24 hours. Purified infected macrophages survived for up to 14 days in culture with progressive growth impairment but little viral replication, showing that the block on lytic gene expression was not merely a delay.

The decisive experiments came when the researchers manipulated the conditions of infection. Raising the infectious dose tenfold, from a multiplicity of one to ten plaque-forming units per cell, transformed the outcome entirely: the frequency of lytic reporter-positive cells jumped from about 4 percent to roughly 92 percent, accompanied by degradation of host actin mRNA, a hallmark of the virus-induced host shutoff mediated by the viral SOX exonuclease, along with viral DNA replication, viral protein production, and release of new infectious virus. Notably, this productive infection came at a steep cost, with high rates of infected cell death. Separately, pre-treating macrophages with the cytokine interleukin-4 before infection at the intermediate dose overcame the restriction, driving approximately 60 percent of cells into lytic gene expression and enabling substantial virus production. This result aligns with earlier work showing that helminth co-infection, which elicits IL-4 and IL-13, can reactivate latent gammaherpesvirus in macrophages through STAT6-dependent activation of the viral RTA promoter.

Viral genetics added a further layer of control. A mutant virus lacking the viral cyclin vCyc, a gene dispensable for lytic replication in fibroblasts but critical for reactivation from latency, showed a greatly reduced frequency of lytic marker expression in macrophages. Conversely, a virus engineered to overexpress Rta, the essential lytic transactivator, dramatically increased the fraction of cells entering the lytic cycle. Together, these findings show that macrophage infection is governed by at least two regulatory barriers: a block on entry into lytic replication that can be overcome by high viral dose or host cytokine signaling, and a cell death pathway activated when productive replication does proceed.

The study’s authors are careful about interpretation. They refer to the dominant restricted state as restricted rather than latent, because while these cells retain viral genomes and express limited viral genes, it remains unknown whether they meet the formal definition of herpesvirus latency, which requires the capacity to resume lytic gene expression and typically evidence of a circularized genome. The in vitro system also relies on a cell line rather than primary macrophage subsets, which are known to respond differently to infection in vivo. Nevertheless, the work fundamentally reframes how scientists think about gammaherpesvirus interactions with myeloid cells. It suggests that bulk analyses averaging across thousands of cells have obscured the true heterogeneity of infection, that the ORF75 locus may play an unappreciated regulatory role in macrophages, and that interventions targeting ORF75-derived T cell epitopes might selectively disrupt macrophage infection. As the authors conclude, the complexity of gammaherpesvirus-myeloid cell interactions is not only real but knowable, and single-cell approaches will be essential to unraveling it.

Subject of Research: Regulation of gammaherpesvirus infection outcomes in macrophages by host and viral factors

Article Title: Gammaherpesvirus infection of myeloid cells reveals divergent outcomes regulated by host and viral factors

Article References: Vragel, G., Noell, K. S., Tseng, A., Kostelecky, R. E., Alexander, E. A., Cohen, S., Galvan, C., Dalwadi, M., Gomez, B. D., Medina, E. M., Oldenburg, D. G., Goodspeed, A., Clambey, E. T., & van Dyk, L. F. (2026). Gammaherpesvirus infection of myeloid cells reveals divergent outcomes regulated by host and viral factors. iScience, 29(11), Article 117872. https://doi.org/10.1016/j.isci.2026.117872

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117872

Keywords: gammaherpesvirus, MHV68, macrophages, single-cell RNA sequencing, lytic replication, latency, ORF75, interleukin-4, viral cyclin, RTA, myeloid cells, herpesvirus pathogenesis