The first translated transcriptional unit of the virus, the N protein, complexes with the gRNA to form the RNP, the template for RNA synthesis. Only encapsidated RNA, not naked
RNA, can be transcribed. RNA synthesis begins with the binding of the RdRp, a complex of the P and L proteins, to the RNP, as inferred from other studies of highly homologous viruses.¹⁴⁰,¹⁴⁷

The MuV N protein is a bipartite molecule consisting of a globular N-terminal assembly domain (amino acids 1 to 398) mediating RNA binding (to form the RNP), and an unstructured hypervariable C-terminal tail believed, based on studies of related viruses, to interact with the MuV M protein during virion assembly.³⁵,⁸⁸,³⁴²

The MuV P protein forms homotrimers via a centrally located coiled-coil domain⁸⁷ and complexes with the L protein to form the RdRp. The C-terminal 48 amino acids of the P protein is a nucleocapsid-binding domain (NBD), responsible for tethering of the RdRp to the RNP by binding to the assembly domain of the N protein (amino acids 1 to 398).²⁰⁷ The three-dimensional structure of the NBD has been deciphered using X-ray crystallography and revealed formation of a compact bundle of three α-helices, a feature that appears to be conserved among paramyxovirinae.²⁰⁸ Unlike the NBD of measles virus and Sendai virus, that of MuV lacks defined tertiary structure and is fundamentally unstable.²⁰⁷ As indicated by its name, the MuV P protein is heavily phosphorylated,²⁷⁸ an action believed to regulate polymerase activity, but this has not been clearly demonstrated for MuV.¹²³,³⁶¹,³⁶²

Six functional domains have been identified in the MuV L protein based on sequence similarity with L proteins from related viruses.³⁰⁸,³⁵⁰ These domains have been ascribed all catalytic functions such as execution of transcription and replication, as well as methylation, capping, and polyadenylation of mRNAs (see Chapter 33). A detailed functional and structural analysis of the MuV L protein has not yet been carried out.

The MuV M protein orchestrates virus assembly and budding. Only the MuV M protein when expressed alone is sufficient for virus-like particle production, although in the absence of co-expression with other viral proteins, efficiency is low.²³⁴ Evidence indicates that the M protein binds to the cytoplasmic tails of the F and HN glycoproteins assembled at distinct locations on cellular membranes, presumably lipid raft microdomains. There, the M protein functions as an adapter, physically linking the region of host-cell membrane expressing the viral F and HN glycoproteins with the viral RNP via its interaction with the N protein.¹⁵²,²³⁴ Budding appears to be mediated by an interaction between the MuV M protein and the cellular endosomal sorting complex for transport (ESCRT) machinery.²³⁴ The MuV M protein has also been shown to interact with host proteins angiomotin-like 1 (AmotL1) and 14-3-3.²⁹⁸,²⁹⁹ Whereas the AmotL1–M protein interaction appears to promote MuV VLP production, based on studies of PIV5, M protein interaction with 14-3-3 decreases the efficiency of virus budding. Interestingly, the MuV M 14-3-3 binding site is adjacent to a sequence motif conserved among rubulaviruses, presumably functioning as a binding site for the host protein caveolin 1 (Cav-1), an essential structural component of caveolae that are considered a subset of lipid rafts. It is therefore likely that MuV budding, like that of PIV5, occurs from caveolae. The close proximity of the 14-3-3 and postulated Cav-1 binding sites raises the possibility that the MuV
M protein switches between either binding to 14-3-3 or Cav-1, thereby regulating the amount of M that can participate in virus budding.²⁹⁹

The F and HN are transmembrane glycoproteins of types I and II, respectively. The F glycoprotein is synthesized as an inactive precursor, F₀, that is targeted to the rough endoplasmic reticulum via a 19 amino acid signal peptide, which is subsequently cleaved.⁴⁰⁸ Following N-glycosylation, the precursor is transported to the trans-Golgi network where it is proteolytically cleaved between amino acids 102 and 103 by the host cell protease furin at the R-R-H-K-R motif to produce two disulfide-linked heterodimers F₁ and F₂.¹⁶²,²⁵⁷,²⁷⁸,⁴⁰⁹ Cleavage of F₀ is essential for virus-to-cell and cell-to-cell membrane fusion and for virus infectivity. The amino terminus of the F₁ subunit possesses the fusion peptide, a conserved hydrophobic domain exposed by the cleavage event (see Chapter 33). Evidence indicates that a second cleavage event occurs during which F₁ is processed into two subunits, F_1a and F_1b; this event is important in mediating fusion activity.⁴⁰⁰

At least two heptad repeat (HR) domains are found in the F1 ectodomain: HR1 at the amino terminus adjacent to the fusion peptide and HR2 at the carboxyl terminus adjacent to the transmembrane domain.²³⁹ The MuV F protein forms homotrimers and the HR1 and HR2 domains interact to form a stable six-helix bundle structure.²³⁹,²⁴⁹ The HR2 domain is also involved in the binding of F with HN. In related viruses, additional HR domains have been identified, although this has not been confirmed for MuV.¹³¹,²⁷⁰ The specific processes involved in MuV fusion have not been delineated; however, based on similarity with the six-helix bundle structural of the PIV5 F protein, the events that mediate MuV fusion are likely similar to those for PIV5 (see Chapter 34).

In its native state, the HN protein is a disulfide-bonded oligomer assembled as homotetramers. The protein is held in the lipid bilayer by a hydrophobic domain of 19 residues near the amino terminal, a domain that probably also serves as a signal sequence in a manner similar to other paramyxovirus HN proteins. HN monomers display a membrane proximal stalk that supports a globular head, forming a six-blade propeller structure.⁸⁴,²¹⁹ The globular head is responsible for attachment and neuraminidase activity¹⁸²,²⁹¹; the stalk region, in conjunction with the F protein, mediates virus-to-cell and cell-to-cell fusion.³⁷¹,³⁸² Based on inference from related viruses, a single site is believed to mediate both neuraminidase and receptor binding activity using a protein conformational switch mechanism to toggle activity.⁷⁷,⁸⁴,²²⁶ There may also be a second sialic acid binding site within the globular head region, theorized to stabilize viral–cellular membrane interactions in addition to the large binding pocket that fluxes between neuraminidase and receptor binding activity.³⁷,³¹⁴,⁴³⁷ Two competing models on the HN-assisted mechanism of F protein activation have emerged⁷⁸ (see Chapter 34 for details). Briefly, the “clamp model” proposes that the HN protein associates with F intracellularly, stabilizing the prefusion conformation of the protein. Upon binding of HN protein to its receptor, the F protein is released, allowing for the conformational change required for fusion activity. In the “provocateur model,” a change in the structure of the HN itself is postulated to promote fusion activity. In this model, the HN protein either is preassociated with the F protein or associates with the protein following receptor binding, at which point the HN protein undergoes a conformational change leading to destabilization of the F protein, which confers fusion activity. Results from recent studies using PIV5 HN and F proteins support the “provocateur model.” Whether this model applies to MuV awaits confirmation.

The MuV V protein, as reported for other rubulaviruses, is involved in inhibiting IFN production and signaling (see Chapter 33). The 69 aa C-terminal cysteine-rich domain of the MuV V protein appears to be the key player in these activities. This region directly interacts with MDA5 (melanoma differentiation–associated gene 5), a pattern recognition receptor that recognizes cytosolic viral RNA, and with the TBK-1 (TANK-binding kinase 1)/IKKε (inhibitor of kB kinase-ε) kinases responsible for interferon regulatory factor-3 (IRF-3) phosphorylation. MuV V protein interaction with MDA5 inhibits its ability to induce transactivation of the IFN-β promoter,¹²,³²⁰ and in the case of TBK-1/IKKε, leads to their ubiquitination and subsequent proteasomal degradation,²⁴³ preventing IRF-3 phosphorylation, an event required for transcription of IFN and IFN-stimulated genes. The C-terminus of the MuV V protein also interacts with the cellular signal transducer and activator of transcription (STAT) proteins, STAT-1, STAT-2, and STAT-3.¹⁵¹,²⁸²,⁴³⁴,⁴³⁵ STAT-1 and STAT-2 play a central role in the IFN signal transduction pathway that eventually leads to activation of IFN-induced genes. STAT 3 has also been implicated in cellular antiviral responses.³¹⁸,³⁸⁹ Binding of MuV V leads to the ubiquitination and subsequent degradation through the proteasomal pathway of STAT-1 and STAT-3, but not of STAT-2; however, the latter is required for targeting of STAT-1 for degradation.²¹⁶,³⁸⁸,³⁸⁹,⁴³⁵ The C-terminus of the MuV V protein is not the only region important in these interactions, as exemplified in a study demonstrating that a single point mutation at amino acid position 95 abrogates the ability of the MuV V protein to degrade STAT3, while retaining the virus’s ability to target STAT 1.³¹⁸

The observation that the MuV V protein can oligomerize and form spherical particles suggests that the MuV protein provides a scaffold for coordinating the assembly of the cellular components (e.g., UV-damaged DNA binding protein 1 [DDB1], cullin 4A [Cul4A], and regulator of cullin 1 [Roc1]) involved in ubiquitination, collectively referred to as V-dependent degradation complexes (VDC).³⁸⁸ For more details on VDC, see Chapter 33.

The SH protein consists of 57 amino acids, 25 of which are highly hydrophobic and clustered at the amino terminus serving as a membrane anchor region with its C-terminus facing the cytoplasm.¹⁰⁴,¹⁰⁶,³⁶⁷ The predicted SH mRNA exhibits two AUG start codons, the second of which represents the actual AUG for the SH open reading frame. The first AUG, located at positions 4 to 6, gives rise to a minicistron with a stop codon at nucleotides 19 to 21.¹⁰⁶,³⁶⁷ Due to the immediate proximity of the first cistron to the cap of the mRNA, it is predicted that with a frequency of about 50%, the ribosomes will skip the first AUG and initiate at the second cistron,²¹² thus enabling translation of the SH protein, although at reduced efficiency. The
biological significance of this minicistron and its proposed role in reducing the amount of SH protein synthesized is unknown.

In certain MuV strains, such as the Enders and Rubini strains, a point mutation exists in the putative F gene polyadenylation signal resulting in an F-SH bicistronic mRNA,²²⁰,³⁶⁷,³⁶⁸ from which only the F protein is made. This demonstrates that the SH protein is not essential for virus replication, which has been confirmed for PIV5.¹⁵⁶

Studies of PIV5 demonstrated that the SH protein facilitates evasion of the host antiviral response via blocking the TNF-α-mediated apoptosis pathway.¹⁵⁶,¹⁵⁷ The MuV SH protein appears to be functionally similar, based on recombinant DNA studies in which PIV5 SH gene was then replaced with that of MuV.⁴¹⁸ Yeast two-hybrid and co-immunoprecipitation studies identified ataxin-1 ubiquitin-like interacting protein (A1Up) as a cellular target of the MuV SH protein. This protein plays a role in proteasomal degradation, but the biological significance of its interaction with the MuV SH protein is not clear.⁴²⁷

Expression of the I protein in infected cells was confirmed, but its role in the life cycle of the virus is unknown.²⁹⁷

Initial clinical symptoms usually relate to infection of the parotid gland, but viral involvement of this gland is neither a primary nor obligate step in the infection.²⁰² Virus infects the ductal epithelium, resulting in desquamation of involved cells, periductal interstitial edema, and a local inflammatory reaction primarily involving lymphocytes. Swelling, inflammation, and tissue damage in the parotid gland can produce elevation of serum and urine amylase levels.³⁴⁶

MuV CNS invasion, as demonstrated by cerebrospinal fluid (CSF) pleocytosis, occurs in greater than one third of patients presenting with clinical mumps.²⁰,⁴²,⁴⁶,¹¹² Symptomatic CNS infection (i.e., meningitis) is less common, occurring in approximately 10% of cases.²⁰,¹⁴⁹,¹⁹⁸ Encephalitis occurs in less than 0.5% of cases. In the prevaccine era, MuV was a leading cause of viral meningitis and encephalitis in most developed countries and continues to be a leading cause in unvaccinated populations worldwide.¹⁸,¹²⁵,¹⁶⁰,²⁵⁸ Neurologic manifestations appear with a 3:1 or greater male–female ratio³⁰,²¹⁰,²²⁸ and are generally preceded by parotitis by 4 to 5 days but can occur before or in the total absence of detectable salivary gland swelling.

As inferred from animal studies, viral invasion of the CNS occurs across the choroid plexus.⁴²⁰ Blood-borne infected mononuclear cells, and possibly cell-free virus, can cross the fenestrated endothelium of the choroid plexus stroma and serve as a source for subsequent infection of the choroidal epithelium. Maturation of virus from the ventricular surfaces of choroidal cells provides progeny virions that are widely distributed through ventricular pathways and the subarachnoid space by CSF (Fig. 35.3). Virus can penetrate brain parenchyma and infect neurons by contiguous spread from infected ependymal cells that line the ventricular cavities of the brain. Once within neurons, virus most likely spreads along neuronal pathways, as reported in nonhuman primates.²³⁰ Although primary encephalitis is typically the response to direct viral invasion of neural cells, cases of postinfectious encephalitis, an autoimmune attack on CNS myelin sheaths, also occurs. Symptoms of primary encephalitis appear before or during the development of parotitis; symptoms of postinfectious encephalitis and associated demyelination appear 1 to 3 weeks after the onset of parotitis.²³⁸,³⁷²

Typical CNS pathology of MuV encephalitis includes edema and congestion throughout the brain with hemorrhage, lymphocytic perivascular infiltration, perivascular gliosis, and demyelination. Findings in the spinal cord include early degenerative changes in the anterior horn cells and perineuronal edema.¹⁰²,²⁶⁴,³⁷² When seen, the selective periventricular myelin loss with relative sparing of axons is typical of parainfectious autoimmune encephalitis.

Rarely is CNS infection fatal and most cases resolve without sequelae. In some instances, however, electroencephalographic changes, ataxia, and behavioral disturbances may take months to resolve¹⁷³,²¹⁰ and permanent neurologic damage—such as obstructive hydrocephalus,²⁸⁶ deafness,²³⁶ and myelitis²⁸⁴,³⁹⁵—can occur.

Hydrocephalus can develop days to years after initial MuV infection and can lead to progressively worsening headaches, mental status changes, and gait abnormalities.⁷¹,¹³⁷,²⁸⁶ The pathogenesis of hydrocephalus is inferred from animal studies that have suggested that desquamation of virus-infected ventricular ependymal cells blocks egress of CSF through the aqueduct of Sylvius. Abnormally restricted flow of CSF to adsorptive sites over the cerebral convexities results in progressive enlargement of the lateral and third ventricles.¹⁹¹,¹⁹² The presence of ependymal cell debris in the CSF of humans presenting with MuV CNS infection suggests a similar mechanism of hydrocephalus induction in humans.¹⁶¹,²⁸⁶,³⁸⁰ Hydrocephalus, however, has also been observed before, or in the total absence of, aqueductal stenosis.¹⁸⁹,³⁶⁴,³⁶⁵,⁴¹⁹ This indicates that stenosis of the aqueduct could be a secondary consequence of external compression by surrounding edematous tissue and not causally related to the pathogenesis of hydrocephalus.

MuV is the most frequent cause of acquired sensorineural hearing loss in children. Transient, high-frequency deafness is the most common form, occurring in approximately 4% of mumps cases.⁴⁰² Permanent deafness occurs in less than 1 per 20,000 cases²⁷,¹⁵³ and is usually unilateral. Deafness is believed to be the result of direct viral invasion of the cochlea, likely via the perilymph, which freely communicates with the CSF¹⁴⁶,²⁵⁴,³⁵²,⁴¹⁵; however, evidence indicates a hematogenous route exists.²⁶⁵ Pathology includes degeneration of the stria vascularis, tectorial membrane, and organ of Corti and collapse of Reissner’s membrane.¹¹¹ Hearing loss caused by indirect effects of virus infection (e.g., immune-mediated damage) have also been suggested.³⁹⁰ Deafness is not disproportionately seen with other complications, suggesting no specific pathogenic link with parotitis, meningitis, or other complications. Deafness has also been observed in otherwise asymptomatic patients.²⁸⁷,³⁹⁰

Orchitis, usually unilateral, occurs in approximately 20% of postpubertal men who develop mumps.¹⁶,¹²⁴,²²⁴ Orchitis rarely occurs in children, suggesting that certain hormonal factors, such as receptors for luteinizing hormone and follicle-stimulating hormone expressed during adolescence, might promote testicular tropism of the virus.³⁷⁶ Virus has been isolated from testicular biopsies of the affected gland within the first 4 days of symptoms, and from semen,²⁹,¹⁷⁹ strongly suggesting that symptomatic gonadal involvement reflects local virus replication. The
seminiferous tubules may be the primary site of viral replication, with local lymphocytic infiltration and edema of interstitial tissues.

Ovary infection is diagnosed in less than 5% of postpubertal women who develop mumps,³⁸,⁶⁴,⁹⁷,¹⁹⁹,²⁴⁸,³⁶⁰ which may be an underestimate because unless a pelvic examination is performed, abdominal or pelvic pain from inflamed ovaries might be attributed to pancreatic infection.

Based on the frequency of viruria, virus frequently disseminates to the kidneys, where epithelial cells of the distal tubules, calyces, and ureters appear to be primary sites of virus replication.⁴¹³ Viruria can be detected in most patients, sometimes for as long as 14 days after the onset of clinical symptoms.³⁹²,³⁹³ Mild abnormalities of renal function have been described, but they are usually of little clinical importance.³⁹² Although virus dissemination to, and replication in, the parotid gland and kidney can occur simultaneously, replication in renal tissue is more prolonged and continues well beyond the appearance of neutralizing antibody in serum.

Pancreatic involvement, diagnosed in 1% to 27% of cases,³⁸,⁴⁷,⁶⁴,²³²,²⁹⁶,³⁴⁵,³⁵⁶,³⁷⁶,⁴¹⁶ is usually expressed as mild epigastric pain, but severe hemorrhagic pancreatitis¹¹⁴ and transient exocrine function abnormalities⁹² have been reported. MuV infects human pancreatic beta cells in vitro,⁴⁰³ and virus infection of the pancreas has been demonstrated in hamsters inoculated intraperitoneally.⁴²⁰ Viral infections have been considered a possible precipitating event leading to the onset of about one third of all cases of juvenile-onset or type I insulin-dependent diabetes mellitus (IDDM); however, whether MuV causes IDDM is unclear.⁸³,¹²⁷,¹³⁶,³⁴⁴ No association has been found between mumps and type II diabetes.²⁴⁰

Myocardial invasion occurs frequently in mumps, as indicated by electrocardiographic abnormalities.¹⁴ Although it is seldom symptomatic, interstitial lymphocytic myocarditis and mild pericarditis may occur following mumps replication in cells of the myocardium and pericardium.⁴³ MuV myocarditis can lead to the rare but serious sequelae of endocardial fibroelastosis.²⁷⁹

Mild to moderately severe mono- or polyarticular and, often, migratory arthritis has rarely been associated with mumps.¹³⁹ MuV has not been isolated from joint fluids or synovial tissues, and no evidence exists for significant immune complex deposition.

MuV can be transmitted transplacentally as demonstrated in nonhuman primates³⁵⁷ and by the isolation of the virus from the human fetus following spontaneous first-trimester abortion during maternal mumps.²²¹,⁴³³ The virus can produce a fetal wastage in humans, with or without subsequent spread of virus to involve fetal tissues directly.⁴³³ MuV has also been isolated from fetal tissues following planned therapeutic abortion of seronegative women 1 week after vaccination with live, attenuated MuV, although it is unclear if the virus detected was vaccine virus, or wild-type virus coincidentally contracted at or shortly before the time of vaccination.⁴³⁰ A proliferative necrotizing villitis with decidual cells containing intracytoplasmic inclusions has been described in the products of spontaneous and induced abortions.¹²⁹ Late-gestation intrauterine infection was reported in an infant born to a mother who developed mumps more than 4 weeks before delivery, diagnosed by reverse transcription polymerase chain reaction (RT-PCR) testing of the infant’s cord blood cells.³⁶³ This infant developed severe pulmonary symptoms, including hypertension and hemorrhage.

MuV is excreted in breast milk,²⁰³ but few cases of perinatal mumps have been described¹⁹⁵,²²² and it is not clear if breast milk was responsible for these cases. There appears to be a somewhat different mode of pathogenesis of mumps in newborns. In the first year or two of life, infants may have only pulmonary involvement without evidence of parotitis.¹⁹⁵,³⁶³ Split immunologic recognition in the infants can follow maternal parotitis, resulting in MuV-specific cell-mediated immune responses without a concomitant antibody response.¹,³⁵⁷,³⁷⁸