Friday, December 9, 2016

UL41: A Weapon of Herpes Simplex Virus 1 Against Innate Immunity

Specialized cells are involved in both
 the innate and adaptive immune systems.
The innate immune system functions as the first line of defense against a viral infection. The system functions in a nonspecific manner, meaning that the recognition of any “non-self” pathogen causes an innate immune response. The innate immune system defends against viral attacks by providing a physical barrier (skin, gastrointestinal tract, hair), inducing an inflammatory response, recruiting specific immune cells to the area of infection, and activating the adaptive immune response. A major part of the innate immune response is the induction of Type-1 Interferon. The interferon response activates by the recognition of viruses by certain cell membrane (Toll-like Receptors) or cytoplasmic (RIG-1, Mda-5, DAI, and AIM2) proteins. The recognition of a virus by these proteins leads to the expression of Type 1 Interferon, which binds to the infected cell’s (autocrine signaling) and neighboring cells’ (paracrine signaling) interferon receptors. The activation of a cell’s interferon receptor leads to the expression of interferon-stimulated genes (ISGs), whose functions include the suppression virus replication and spread, activation Natural Killer cells, and induction of apoptosis (programmed cell death).
Tetratricopeptide repeats are unique structures
 found within IFIT proteins.
Although most Interferon Stimulated Genes remain uncharacterized, scientists have currently identified about 2000 ISGs in humans and mice (1). The IFIT (IFN-induced protein with tetratricopeptide repeats) protein family, including proteins IFIT1, IFIT2, IFIT3, and IFIT5 , have been recent ISGs of interest among researchers of innate immunity (2). The IFIT family of proteins contains unique helix-turn-helix structural regions, termed tetratricopeptide repeats (3) (See Image to Right). The IFIT3 protein has been demonstrated to restrict viral replication in a number of viruses, including hepatitis B virus, human papilloma virus, West Nile virus, Dengue virus, and vesicular stomatitis virus (4, 5, 6). IFIT3 impedes virus replication by binding to viral RNAs, disrupting viral protein synthesis, and interacting with various viral proteins (7).
Zhangtao Jian, Chenhe Su, and Chunfu Zheng from the Institute of Biology and Medical Sciences at Soochow University of Suzhou, China, recently published an article in the Journal of Virology about IFIT3’s interactions with Herpes Simplex Virus 1. Human Herpes Simplex Virus 1 (HSV-1), more generally termed “herpes,” is notorious for causing humiliating blisters (which eventually turn into sores) around the mouth, genitalia, and rectum (8). HSV-1 spreads easily through skin sores or oral secretions, allowing the virus to readily transmit from human-to-human by kissing or sharing drinks (8). Herpesvirus has the ability to establish latency, meaning that symptoms of HSV-1 infection seem to disappear until a period of stress or weakness of the immune system triggers a sudden reactivation of the symptoms. There are no vaccinations or cures for HSV-1 infection; however, antiviral drugs such as Famvir, Zovirax, and Valtrex are used to treat the symptoms (8).
Herpesvirus Simplex 1 has the capability to easily spread from cell-to-cell and avoid host immune defense mechanisms. The large double-stranded DNA genome encodes 80 proteins, many more than your average virus. These proteins play a role in almost every virus function, including virus replication and innate immune response evasion.  The UL41 protein of HSV-1 has been shown to play a role in the virus’s ability to evade the interferon response (9). The UL41 viral protein is able to quickly degrade the host cell’s mRNA, thus preventing cellular synthesis of antiviral proteins that attempt to terminate HSV-1 replication and spread (10, 11). In their study, Jian et al. demonstrate the interactions of the HSV-1 UL41 protein with the interferon-stimulated gene, IFIT3.
Jian et. al. began their experiments by infecting Human Embryonic Kidney 293T (HEK293T) with HSV-1 virus, and adding exogenous (external) IFIT3 to the infected cells, in order study the effects of IFIT3 on HSV-1 replication. The authors used a plaque assay in order to determine the concentration of HSV-1 virus after an eight-hour infection, discovering that exogenous IFIT3 expression did not influence HSV-1 replication levels. Furthermore, western blot analysis, a method of identifying protein levels in cells, demonstrated that HSV-1 infection decreased the amount of IFIT3 compared to uninfected cells. Other IFIT family protein members (IFIT1 and IFIT2) were not affected by HSV-1 infection, suggesting that UL41 targets only IFIT3. Next, the researchers studied if the HSV-1 protein UL41 is responsible for the inability of IFIT3 to inhibit HSV-1 replication and the decrease in overall IFIT3 protein levels. The authors inserted increasing levels of HSV-1 UL41 protein into cells and instead of adding exogenous IFIT3, the authors infected cells with Sendai virus (SeV), which potently induces the expression of cellular IFIT3. The more UL41 viral protein added to the cells, the less IFIT3 protein remained after SeV infection. Furthermore, using RT-qPCR to measure levels of IFIT3 mRNA, the authors discovered that increasing levels UL41 proportionally decreased levels of IFIT3 mRNA in SeV infected cells, suggesting that UL41 degrades IFIT3 mRNA.
Figure 1B: Exogenous IFIT3 expression does not influence concentrations of HSV-1 after eight hours of infection.

Because the authors were still not completely positive that UL41 directly targets UL41 mRNA for degradation, they studied cell infection with R2621, an HSV-1 virus without the UL41 protein.  The authors co-infected cells with SeV and either HSV-1 or R2621 in order to determine if the absence of UL41 increased IFIT3 levels after infection. The co-infection of SeV and HSV-1 resulted in much lower levels of IFIT3 protein and mRNA compared to co-infection with SeV and R2621, strengthening the notion that IFIT3 expression is suppressed independently by UL41. Finally, the authors used the R2621 virus to infect cells with and without exogenous IFIT3, finding that R2621 virus concentrations after infection decreased in cells with exogenous IFIT3. R2621 virus concentration after infection was enhanced by eliminating IFIT3 from cells (by siRNA), confirming IFIT3’s role in inhibiting HSV-1 infection without the presence of the U41 protein. The results of HSV-1 infection with (HSV-1) and without (R2621) the UL41 protein strengthen the notion that UL41 assists in HSV-1 interferon evasion by degrading IFIT3 mRNA.
This study demonstrates Herpes Simplex Virus-1’s ability to evade the innate immune system by UL41’s ability to degrade antiviral IFIT3 mRNA. These results provide only one method of how HSV-1 is able to evade the Type 1 Interferon response. IFIT3 is one of many Interferon Stimulated Genes upregulated by the activation of the interferon pathway. It is possible that many other ISGs are regulated by HSV-1’s UL41 protein’s ability to degrade mRNA. Researchers could perform a similar workflow of experiments used in this study with a variety of other known ISG’s in order to determine a list of proteins involved in the interferon pathway that HSV-1 UL41 targets for degradation. Such future results will provide a greater understanding of the intricate host cell-virus interactions that allow Herpes Simplex Virus 1 to readily infect a host.

Jian Z, Su C, Zheng C. 2016. Herpes simplex virus 1 tegument protein UL41 counteracts IFIT3 antiviral innate immunity. J Virol 90:11056-11061. Doi: 10.1128/JVI.01672-16.

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Figure 1B from Paper

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