Deletion of Murine Gammaherpesvirus Gene M2 in Activation-Induced Cytidine Deaminase-Expressing B Cells Impairs Host Colonization and Viral Reactivation.

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4.

The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4.IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs.

Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

Reassessment of Evidence about Coinfection of Chickenpox and Monkeypox (Mpox) in African Children.

In west and central Africa, monkeypox occurs mainly in older children, adolescents and young adults. In two large epidemiology studies of monkeypox outbreaks, the investigators observed a sizable number of coinfections of chickenpox (varicella) and monkeypox. Based on a review of the literature, we propose that chickenpox (human herpesvirus-3 infection) is a risk factor for acquisition of monkeypox infection. Our hypothesis states that the chickenpox skin lesion provides an entry site for the monkeypox virus, which is harbored on a fomite in the environment of the patient. The fact that monkeypox can enter via a scratch or abrasion is a known mechanism of spread for three other poxviruses, including mousepox (ectromelia), orf and molluscum contagiosum. There are many similarities in pathogenesis between certain poxviruses and chickenpox, including a viremia with a cellular stress response leading to high levels of the IL-6 cytokine. One very revealing observation in the two epidemiology studies was that the number of pox as well as the severity of disease in children with chickenpox and monkeypox coinfection was not greater than found in children with monkeypox alone. Based on the above observations, we conclude that, when chickenpox precedes monkeypox, priming of the immune system by the earlier chickenpox infection moderates the severity of the secondary infection with monkeypox. This conclusion also has important public health implications about chickenpox surveillance.

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome.

IFN-λ Decreases Murid Herpesvirus-4 Infection of the Olfactory Epithelium but Fails to Prevent Virus Reactivation in the Vaginal Mucosa.

Murid herpesvirus-4 (MuHV-4), a natural gammaherpesvirus of rodents, can infect the mouse through the nasal mucosa, where it targets sustentacular cells and olfactory neurons in the olfactory epithelium before it propagates to myeloid cells and then to B cells in lymphoid tissues. After establishment of latency in B cells, viral reactivation occurs in the genital tract in 80% of female mice, which can lead to spontaneous sexual transmission to co-housed males. Interferon-lambda (IFN-λ) is a key player of the innate immune response at mucosal surfaces and is believed to limit the transmission of numerous viruses by acting on epithelial cells. We used in vivo plasmid-mediated IFN-λ expression to assess whether IFN-λ could prophylactically limit MuHV-4 infection in the olfactory and vaginal mucosae. In vitro, IFN-λ decreased MuHV-4 infection in cells that overexpressed IFN-λ receptor 1 (IFNLR1). In vivo, prophylactic IFN-λ expression decreased infection of the olfactory epithelium but did not prevent virus propagation to downstream organs, such as the spleen where the virus establishes latency. In the olfactory epithelium, sustentacular cells readily responded to IFN-λ. In contrast, olfactory neurons did not respond to IFN-λ, thus, likely allowing viral entry. In the female genital tract, columnar epithelial cells strongly responded to IFN-λ, as did most vaginal epithelial cells, although with some variation from mouse to mouse. IFN-λ expression, however, failed to prevent virus reactivation in the vaginal mucosa. In conclusion, IFN-λ decreased MuHV-4 replication in the upper respiratory epithelium, likely by protecting the sustentacular epithelial cells, but it did not protect olfactory neurons and failed to block virus reactivation in the genital mucosa.