Cytomegalovirus-Specific T Cell Epitope Recognition in Congenital Cytomegalovirus Mother-Infant Pairs.

Background: Congenital cytomegalovirus (cCMV) infection is the most common infection acquired before birth and from which about 20% of infants develop permanent neurodevelopmental effects regardless of presence or absence of symptoms at birth. Viral escape from host immune control may be a mechanism of CMV transmission and infant disease severity. We sought to identify and compare CMV epitopes recognized by mother-infant pairs. We also hypothesized that if immune escape were occurring, then one pattern of longitudinal CD8 T cell responses restricted by shared HLA alleles would be maternal loss (by viral escape) and infant gain (by viral reversion to wildtype) of CMV epitope recognition. Methods: The study population consisted of 6 women with primary CMV infection during pregnancy and their infants with cCMV infection. CMV UL83 and UL123 peptides with known or predicted restriction by maternal MHC class I alleles were identified, and a subset was selected for testing based on several criteria. Maternal or infant cells were stimulated with CMV peptides in the IFN-γ ELISpot assay. Results: Overall, 14 of 25 (56%; 8 UL83 and 6 UL123) peptides recognized by mother-infant pairs were not previously reported as CD8 T cell epitopes. Of three pairs with longitudinal samples, one showed maternal loss and infant gain of responses to a CMV epitope restricted by a shared HLA allele. Conclusions: CD8 T cell responses to multiple novel CMV epitopes were identified, particularly in infants. Moreover, the hypothesized pattern of CMV immune escape was observed in one mother-infant pair. These findings emphasize that knowledge of paired CMV epitope recognition allows exploration of viral immune escape that may operate within the maternal-fetal system. Our work provides rationale for future studies of this potential mechanism of CMV transmission during pregnancy or clinical outcomes of infants with cCMV infection.

Impact of dynamin 2 on adenovirus nuclear entry.

The large GTPase dynamin 2 controls both endosomal fission and microtubule acetylation. Here we report that dynamin 2 alters microtubules and regulates the trafficking of human adenovirus type 37. Dynamin 2 knockdown by siRNA in infected cells resulted in accumulation of acetylated tubulin, repositioning of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (with a compensatory decrease in endosomal virus), reduced proinflammatory cytokine induction, and increased binding of virus to the nucleoporin, Nup358. These events led to increased viral DNA nuclear entry and viral replication. Overexpression of dynamin 2 generated opposite effects. Therefore, dynamin 2 inhibits adenovirus replication and promotes innate immune responses by the infected cell. MTOC transposition in dynamin 2 knockdown promotes a closer association with nuclear pore complexes to facilitate viral DNA delivery. Dynamin 2 plays a key role in adenoviral trafficking and influences host responses to infection.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection.

Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5' to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5'-to-3' exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E-DNA-ddNTP) and binary (E-DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5'-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.