Elucidating the Roles of Adaptative Polymorphisms in Modulating the Inhibitory Effects of RL13 on Human Cytomegalovirus Replication

Human cytomegalovirus (CMV), a member of the herpesvirus family, causes significant disease in immunocompromised patients and is the major infectious cause of birth defects when acquired congenitally. When CMVs in clinical samples are propagated in cell culture they rapidly acquire substitutions, frameshifts, or deletions in the RL13 gene that result in increased release of infectious virions into the culture medium. RL13 encodes RL13, a glycoprotein found in the virion envelope. How or why RL13 restricts release of cell-free virus is not known. In previous work we found that propagation of CMV in the presence of CMV-hyperimmunoglobulin (HIG) prevents mutations in RL13. However, extended passage with HIG gave rise to a virus designated Ig-KG-H2 containing additional mutations resulting four amino acid substitutions in glycoprotein M, an envelope glycoprotein involved in virion entry, two amino acid substitutions in the UL102 helicase primase subunit, and one amino acid substitution in the viral immediate early 2 (IE2) protein. As RL13 mutated slowly when Ig-KG-H2 was passaged without HIG, we hypothesized that one or more of the mutations impacting gM, UL102, or IE2 may act to modulate the inhibitory effects of RL13. In Aim 1 laboratory CMV strains derived from infectious bacterial artificial chromosome (BAC) clones were modified to encode RL13 fused to a C-terminal FLAG epitope. Small focus size and mutation of the RL13FLAG gene confirmed that RL13FLAG retains the growth impairment functions of RL13. Western blotting using a-FLAG antibody confirmed RL13FLAG expression and immunofluorescence localized RL13FLAG to large perinuclear structures that likely correspond to virion assembly compartments where virion morphogenesis occurs. In Aim 2, genetic swaps introducing sequences encoding gM from Ig- KG-H2 into two laboratory CMV strains resulted in increased focus size and preservation of RL13 during serial passage, indicating that polymorphisms in gM that arose during passage of Ig-KG-H2 with HIG function to antagonize the inhibitory effects of RL13. In Aim 3 a single H390D substitution in IE2 was shown to decrease RL13FLAG levels, increase focus size, and preserve of RL13 during serial passage, confirming that the IE2D390 polymorphism antagonizes the inhibitory effects of RL13. The same IE2D390 polymorphism was previously shown by others to permit CMV replication in the absence of UL84, a viral protein normally required for DNA replication. These results suggest two potentially independent mechanisms by which RL13 impairs CMV replication. First, interactions between RL13 and gM in vesicular membranes or within virion envelopes may alter virion morphogenesis within virion assembly compartments to promote cell-to-cell spread and reduce cell-free virus release. Second, direct or indirect interplay between RL13 and viral proteins involved in DNA replication (IE2, UL84, and perhaps UL102) may modulate the efficiency of viral DNA replication. Why CMV has evolved mechanisms to impair its replication and restrict release of cell-free virus remains uncertain; however, that in vivo CMV persists long-term in the presence of extracellular neutralizing antibodies suggests that RL13 may function to reduce unproductive release of cell-free virions since in vivo extracellular virions would be rapidly neutralized. This further suggests that in certain anatomical sites, such as kidneys or salivary glands, unknown mechanisms may actively antagonize RL13 in order to promote shedding of cell-free virions into urine or saliva and thereby transmit the infection to new hosts