APOBECs and Herpesviruses

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded (ss)DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes-APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new pathogen-host conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

ABSTRACT Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129 , which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130 , an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10 6 -fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10 6 PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCEHuman cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection

Essential Role for either TRS1 or IRS1 in Human Cytomegalovirus Replication▿ †

Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2′-5′ oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[ΔI/ΔT]) has a severe replication defect. Infection with HCMV[ΔI/ΔT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[ΔI/ΔT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2α is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection

Assessing Zika virus replication and the development of Zika-specific antibodies after a mid-gestation viral challenge in guinea pigs.

Primary Zika virus (ZIKV) infections that occur during pregnancy can cause spontaneous abortion and profoundly disrupt fetal development. While the full range of developmental abnormalities associated with congenital Zika syndrome is not yet known, severe cases of the syndrome can present with microcephaly, extensive neurologic and ocular damage, and pronounced joint malformations. Animal models that accurately recapitulate congenital Zika syndrome are urgently needed for vaccine development and for the study of ZIKV pathogenesis. As guinea pigs have successfully been used to model transplacental infections by cytomegalovirus, syphilis, and Listeria monocytogenes, we sought to test whether ZIKV could productively infect guinea pigs and whether viral transmission with attendant fetal pathology would occur after a mid-gestation viral challenge. We found that guinea pig cells supported ZIKV replication in vitro. Experimental infection of non-pregnant animals did not result in overt disease but low-level, detectable viremia was observed. When pregnant guinea pigs were challenged with ZIKV at between 18 and 21 days gestational age, ZIKV was not detected in maternal or pup blood, plasma, or tissues and no significant differences in maternal weight gain or pup size were observed following challenge. Nonetheless, a robust antibody response against ZIKV was detected in both the pups and dams. These results suggest that, while guinea pigs can model aspects of the immune response to ZIKV infection during pregnancy, naturally circulating ZIKV strains are not pathogenic during the pregnancy of immunocompetent guinea pigs and do not interfere with normal pup development

APOBECs and Herpesviruses

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded (ss)DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes—APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new pathogen-host conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection

ZIKV infection of pregnant guinea pigs.

<p>Pregnant guinea pigs were infected with 1X10⁷ PFU of ZIKV (N = 21) or sham injected (N = 15) at GA 18–21. <b>(A)</b> Guinea pigs were weighed daily until a pre-determined experimental endpoint and the mean weight change for each group was plotted along with the standard error. Animals were euthanized at 10, 20, 30 or 40 days post-infection (N = 4 infected, 3 uninfected per group) or euthanized on the day of delivery (N = 5 infected, 3 uninfected per group). <b>(B-F)</b> Pups were weighed at the indicated endpoint post-infection. Symbol shape represents individual litters; open symbols indicate pups born dead. The bars indicate the standard error within each group.</p

Evolution of the anti-NS1 response in pregnant dams.

<p>The IgG response to ZIKV NS1 of pregnant guinea pigs and their pups was measured by ELISA. <b>(A)</b> The mean anti-NS1 titer of pregnant guinea pigs challenged with ZIKV (N = 5) or sham injected (N = 2) was measured over time and plotted; bars indicate the standard error for each time point. <b>(B)</b> The anti-NS1 titers of pregnant dams and pups were measured at each experimental endpoint. The bars indicate the mean antibody titer at each time point.</p

Anti-ZIKV antibody production by infected guinea pigs.

<p>To assess whether an antibody response to ZIKV proteins is elicited in challenged guinea pigs, purified ZIKV Env and NS1 and semi-purified viral particles were resolved by SDS-PAGE, transferred to membranes, and immunoblotted. <b>(A)</b> Coomassie stained gel showing the sizes of input protein. <b>(B-E)</b> Proteins were transferred to membranes and immunoblotted using plasma collected from four ZIKV challenged pregnant guinea pigs either the day before viral challenge (D-1) or the day of delivery (Term).</p